Patent Application
20220074831
The present technology relates to methods and apparatus that selectively extract constituents from biological samples for further analysis or diagnostic purposes including, for example, tangential flow extraction, single stage extraction and multiple stage extraction, using separation mechanisms such as membranes. Certain exemplary embodiments of the extraction technology may involve bandpass filtering using tangential flow filtering/extraction to separate components of biological samples such as blood samples (diluted or undiluted), positive blood cultures, urine samples, etc. which may facilitate direct and rapid identification and/or antimicrobial susceptibility testing.
Biological samples (i.e. samples pertaining to life or living organisms) are analyzed in a multitude of contexts. Biological samples are complex mixtures of cells, proteins, vesicles and other components. These components vary among themselves in terms of structure, size, composition, etc. It is often desirable to selectively analyze discrete components of biological samples for research, diagnosis, epidemiology, treatment, etc. For example, the presence or absence of bacterial or fungal pathogens in biological samples can be used to diagnose conditions such as sepsis or urinary tract infections in a patient/subject. As another example, the type and distribution of extracellular vesicles in a sample component (e.g., cells) may provide information useful in the detection and treatment of cancer. However, detection and analysis of discrete components (e.g. pathogens, cells, or extracellular vessels) in biological samples such as blood is complicated by the presence of the other components in the biological samples, which may interfere with the methods designed to detect a component of interest. The ability to isolate and/or concentrate the component of interest and separate it from other components of the biological sample is therefore of critical importance.
Methods for such isolation exist but such methods are not completely desirable. For example, centrifugation and wash methods are labor-intensive and subject to user-variability. Filtration methods have tendency to lose the component of interest to interactions with the filter surface(s). Microbiology plating methods allow the component of interest (e.g. bacteria) to be grown and separated, but require long replication times.
Methods of isolation/concentration that require long wait times are particularly problematic. One important application where there is a need for improved sample processing methods is sepsis detection. Patients admitted to an Intensive Care Unit (ICU) with severe sepsis have a 39.8% risk of death. Therefore, rapidly identifying the responsible pathogen and appropriate antibiotic treatment is imperative for improving patient survivability. Current methods for determining the identity (ID) and antimicrobial susceptibility testing (AST) of pathogens recovered from a positive blood culture (PBC) require the specimen to be isolated from the blood background. The standard approach is to subculture an aliquot of the PBC to isolate and, in some embodiments, further concentrate the pathogens. This subculturing step can delay the time to results by approximately 18-24 hours.
Therefore, it is desirable to implement improved techniques and devices for evaluating components of interest in biological samples that can isolate and evaluate such components quickly. Such techniques may improve the efficiency and/or accuracy of testing.
Examples of the technology disclosed herein can provide a process that may effectively separates pathogens from the blood background without subculturing and may thereby facilitate rapid ID/AST. The sample preparation may include: i) separation of the target pathogen from the sample (e.g. isolation due to size exclusion); ii) increasing the concentration of the target pathogen; or iii) purification of the target pathogen by washing out the impurities while retaining the target. In one example, the target pathogen is a bacterium that is isolated and collected for identification testing or susceptibility testing. Such testing can be paired with automated analysis such as digital imaging of plated cultures over time for the detection of microbial growth thereon or light testing of target pathogen cultures.
Another aspect of the present technology is to implement extraction of components of a biological sample for concentrating a desired constituent.
Another aspect of the present technology is to facilitate biological sample analysis without requiring a subculture step to obtain a sample with a sufficient amount of biomass/colony mass and/or with sufficient purity.
In one embodiment, an apparatus for extracting constituents from a biological sample for concentrating a desired constituent of the biological sample includes a plurality of membrane stages, wherein each of the plurality of membrane stages has a membrane having a first side, a second side, a first end and a second end, the membrane having a characteristic to selectively permit one or more constituents of the biological sample to pass through the membrane from the first side to the second side while retaining other constituents of the biological sample at the first side. The membrane also has an input chamber having an inlet proximate to the first end and an outlet proximate to the second end, the input chamber configured at the first side of the membrane to permit (a) a tangential flow of the biological sample along a first surface of the membrane at the first side from the inlet to the outlet, and (b) a trans-membrane passing of the one or more constituents of the biological sample from the first side to the second side. The apparatus also has an output chamber configured at a second surface of the membrane at the second side and configured to receive the one or more constituents of the biological sample that pass through the membrane. The membrane stages are collectively configured to isolate a desired passband of constituents of the biological sample.
In another embodiment, there is a single stage tangential flow membrane device that has an input chamber having first and second outlets. The device also has an output chamber having a second outlet. The filtration membrane defines a tangential flow path that separates the first chamber from the second chamber and also has a recirculating flow path for directing the sample to pass over the filtration membrane a plurality of times. A component of interest is recovered from the portion of the sample that is retained by the filtration membrane in the input chamber (i.e. the retentate).
In another embodiment, the single stage tangential flow membrane device is configured as a cartridge. The cartridge has a port that receives a sample container, and the port is fluidically coupled to a sample chamber in the cartridge. The sample chamber is fluidically coupled to the first inlet of the input chamber. The first outlet of the input chamber is fluidically coupled to the sample chamber such that retentate from the filtration membrane is recirculated back to the sample chamber.
A method for extracting constituents from a biological sample is described herein. According to the method, in a first extracting process, one or more constituents of the biological sample are passed through, by a pressure force, a first membrane from a first side of the first membrane to a second side of the first membrane while retaining other constituents of the biological sample at the first side of the first membrane and while generating a tangential flow of the biological sample along a surface of the first membrane at the first side of the first membrane. In a second extracting processes one or more additional constituents of the one or more constituents are passed through, using a pressure source, a second membrane from a first side of the second membrane to a second side of the second membrane while retaining some of the one or more constituents at the first side of the second membrane and while generating a tangential flow of the one or more constituents along a surface of the second membrane at the first side of the second membrane. According to the method the first extracting process and the second extracting process isolate, purify and/or concentrate a desired passband of constituents of the biological sample from other constituents of the biological sample.
In another embodiment, a method for isolating pathogens from a positive blood culture is disclosed. According to the method, a blood culture suspected to contain target pathogens is flown into a sample chamber. The blood culture is flown into a filtration device. The filtration device comprises a filtration membrane that separates a first chamber from a second chamber. The blood culture is flown tangentially across the filtration membrane such that the target pathogens, if present in the positive blood culture, flow across the filtration membrane and through a first outlet of the first chamber as retentate. Portions of the positive blood culture having a particle size less than a predetermined threshold flow through the filtration membrane and into the second chamber. The retentate is flown through the first outlet of the first chamber to recirculate over the filtration membrane whereby a further portion of the sample permeates through the filtration membrane. After recirculating the retentate a predetermined number of times, the retentate is washed by combining the retentate with a wash buffer. The washed retentate in a vial for downstream testing to determine the presence or absence of target pathogens in the retentate.
Other features of the technology will be apparent from consideration of the information contained in the following detailed description, drawings and claims.
The present technology is illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings, in which like reference numerals refer to similar elements including:
Some embodiments of the present biological extraction technologies may implement employing tangential flow extraction employing one or more extraction stages.
Tangential Flow Extraction Principle and Concept
The present technology provides apparatus and methods to isolate or purify (and in some embodiments concentrate) particular constituents of interests from biological samples including, for example, blood, urine, saliva and other sample types using membrane-based technologies in combination with tangential flow extraction. Examples of particular constituents of interest can be target analytes, dissociated or circulating tumor cells (CTC), white blood cells (WBC), extracellular vesicles like exosomes, yeast, bacteria and virus, etc. The membrane-based technology can be implemented with tangential flow processing of a biological sample in liquid form. Such a tangential fluid flow may pass (e.g., repeatedly) the sample over a specified membrane surface that has a trans-membrane pressure characteristic that is engineered to allow a target constituent to pass through the membrane as a permeate or to be retained by the membrane as a retentate. Thus, the specified membrane or membrane device may be selectively configured depending on the target and whether the target will be passed or retained. This technique, unlike many other technologies, offers a beneficial opportunity to maintain viability and integrity of vulnerable extracted constituents such as cells, bacteria, virus, etc. that can be compromised by harsh extraction conditions. In addition, tangential flow separation described herein permits efficient recovery of components extracted from the sample, even at low concentrations, because exposure of the components to the membrane surface area is significantly reduced compared to other filtration approaches such as dead-end filtration or pass-through filtration in which the filter is made of fibrous materials. Thus, the separation method and device described herein reduces the risk of target loss due to binding or damage.
In order to achieve extraction of the target constituents, device design, membrane pore size and process parameters are engineered. The target constituent can be retained or passed through the device membrane depending on the objectives of the extraction process. The porous membrane is preferred to have a generally uniform pore size (a characteristic pore size) and smooth surface relative to the particles to be extracted. One such type of membrane is a track etched membrane (TEM) which can be made from polyester, polypropylene, polycarbonate, polyimide etc. Materials suitable for TEMs are well known to those skilled in the art and not described in detail herein. Such membranes are commercially available and can have uniform pore size ranging from nanometer to micrometer size. Additionally, such membranes can be further treated to modify surface properties such as hydrophobicity to reduce membrane fouling or otherwise improve application performance. Such coatings and treatments are well known to the skilled person and not described in detail herein. The system may preferably be designed to have a correlated tangential flow rate and trans-membrane pressure. The tangential flow rate and trans-membrane pressure are chosen to: i) prevent fouling of the membrane due to phenomena such as cake layer formation or pore clogging with particulate; and ii) optimize the sample processing time without introducing undesired shear or lysing of cellular component. Such flow rate and pressure characteristic selection can allow the target particle size to stay in the retentate phase or pass through the selected membrane as the permeate phase when paired with the proper selection of the membrane pore size for the target constituents to be extracted.
An extraction process is illustrated with reference to the device depicted in
Optionally, the tangential flow (i.e., along the membrane surface) and/or trans-membrane flow (i.e., across the membrane) can be driven by a pressure differential; i.e., by either a positive pressure source and/or a negative pressure source (e.g., a vacuum source). The tangential flow can be one directional flow or reciprocating flow (where fluid flow reverses its direction in order to allow fluid flow over the same membrane surface multiple times to complete the filtration process). Multiple passes of the sample over the membrane provides a number of potential benefits including the reduction of the required membrane area. In some implementations, the tangential flow can be powered by a circulation or pressure pump for driving tangential flow in one direction. In some such cases, the sample will flow multiple times along the surface of the membrane such as where the flow path employs a loop or circuit and a pump circulates or re-circulates the sample fluid in the loop/circuit. In other examples, the sample flows multiple times in different directions along the surface of the membrane such as where the flow path is terminated (e.g., non-looped) and it is coupled to one reversible pump (i.e., bi-directional) or a plurality of alternating uni-directional pumps that achieve the reciprocating tangential flow. In some examples, such pumps/vacuums may optionally be implemented by one or more syringes, which may be manually or automatically operated. Other types of pumps/vacuums may optionally be implemented, or appropriate valving configurations using a single power source deployed, thereby achieving the desired flow. Pumps/vacuums that can be used to drive the flow of the sample across the membrane are well known to one skilled in the art and not described in detail herein.
In some versions, a pressurized container and/or depressurized container can be coupled to the inlet/outlets (101, 103) of the chamber(s) to provide the force for the tangential flow. For example, such a coupling may be at or near opposing ends of the membrane to induce the tangential flow along one surface of the membrane from end to end (i.e., in one chamber). The chamber can be configured to induce flow along the membrane such as when the chamber is bounded by a wall structure 107 to form a narrow gap proximate to and along the surface of the membrane. The tangential flow occurs within the gap. Such as gap may beneficially be longer in dimension along the length (L) of the membrane when compared to the depth (D) of the gap between the membrane and a surface of the wall structure 107 that forms the gap.
As illustrated in
Alternatively, the tangential flow can also be created by moving the membrane. One such example is illustrated in
In the example of
As previously mentioned, the membrane device 100 can be implemented for a single stage extraction such as where the sample tangentially flows along a single membrane or multiple membranes with the same filter characteristics (e.g., same pore size). Such a process may be implemented for bacteria recovery and concentration from fresh whole blood, urine, cerebrospinal fluid, saliva, respiratory samples, and wounds, as well as other sample types. Example processes are described herein.
(1) Bacteria Recovery and Concentration from Fresh Whole Blood
As previously mentioned, fast results of bacteria identification/detection and antibiotics susceptibility testing are desired to improve treatment efficiency. Conventional test procedures involve a long incubation time and complex sample purification steps due to low bacteria concentration in the original samples such as blood or urine. Concentrating the bacteria directly from the patient sample delivers quicker results by improving the detectability or reducing the incubation time.
Tangential flow extraction can serve as a concentration process using a membrane device 100 and can achieve the enrichment of the bacteria in the original sample, such as in an E. coli bacteria recovery test. For example, bacteria may be isolated and, in some embodiments, concentrated from a blood sample. In an example of such a process as illustrated in
In one example, a tangential flow filtration system is similar to the membrane device 600 with a first chamber geometry of 10 mm×40 mm×0.08 mm. The membrane pore size of 0.4 um was used. The 10 mL diluted blood sample (2 mL whole blood and 8 mL BACTEC media) containing spiked bacteria (E. coli) were processed at the tangential flow rate of 10 mL/min with a trans-membrane pressure of approximately 3 psi in a reciprocating flow mode where the fluid flow reverses flow direction multiple times along the membrane surface. The process was stopped when the retentate volume reached to about 0.5 mL and the retentate was collected for further bacteria count determination by plating on an eosin-methylene blue (EMB) plate and incubating overnight. Table 1 below shows that the bacteria viability was not affected by the reciprocating multi-pass tangential flow filtration process and showed good recovery. The percent average recovery is calculated based on average recovered bacteria CFU count from TEM devices as referenced to the count from the sample taken directly from the differentially lysed blood solution (centrifuging the blood to collect the bacteria). About 95 to 100% recovery is achieved in this study for bacteria concentration above 03 CFU/mL. At the low spike concentration of 5 CFU/mL input whole blood, the recovery is 82% on average. The loss can be attributed to the loss of bacteria that occurs in the device due to the dead volume or due to a minor loss of viability of a few microorganisms. The loss of just a few organisms in the low spike results in a greater measured loss since each one represents a higher percentage of the total than those in the higher titer samples.
a Bacteria concentration is based on whole blood volume. Control sample is taken from blood culture bottle. Results are the average of three plate counts.
bConcentrate was plated on three EMB plates per device.
cConcentrate was plated on one EMB plate per device.
(2) Bacteria Recovery and Isolation from a Positive Blood Culture
As previously described, rapid bacteria identification and antibiotic susceptibility testing of bacteria are sought to improve treatment efficiency. Conventional test procedures involve a lengthy subculture step after the identification of a positive blood culture to obtain bacteria for identification and antibiotic susceptibility testing. Isolating and washing the bacteria directly from the positive blood culture sample delivers quicker results by reducing the need for a lengthy subculture step. Antibiotic susceptibility testing and identification testing via MALDI-TOF requires a clean bacteria sample. Background components from the blood culture such as cells, cellular debris, salts, proteins and others need to be removed in order to have reliable test results. In addition, the bacteria concentration in the output sample needs to be sufficiently high so that enough biomass is available (approximately 108 CFU/mL) for ID by MALDI-TOF). The benefits of rapid identification (e.g., via MALDI-TOF) and antibiotic susceptibility testing (e.g. by microbiological or biochemical methods) are increased by quickly isolating the bacteria directly from a blood culture and avoiding the subculture step used widely today.
Tangential flow extraction can serve to isolate bacteria from a sample using the membrane device 100. For example, in one process, bacteria may be isolated from a positive blood culture. In an example of such a process, 10 mL patient blood sample is collected into a BACTEC bottle, and inoculated with bacteria such as E. coli, S. aureus, S. pneumoniae, or other bacteria. The sample is incubated in a BACTEC blood culture instrument until a positive blood culture is detected. Within 0-8 hours from detection, the positive blood culture is applied to a tangential flow extraction process where bacteria will be isolated and washed. Referring again to
The positive blood culture sample flows (repeatedly in a multi-pass process) along the track etched membrane in membrane device 600 in the first chamber (e.g., upper chamber; 104 in
The success of a tangential flow filtration using a membrane device 600 depends on several interrelated parameters of a filtration system selected for a target constituent. In this example, the target constituent is bacteria in positive blood culture. The critical parameters include membrane pore size, chamber geometry, tangential flow rate, and trans-membrane pressure. In addition, the sample to be processed may have variations in solid content, particle size distribution and concentration, viscosity, shear sensitivity, propensity to adhere to surfaces, degree of biofouling, coagulation etc. Highly efficient recovery of a range of potentially different bacteria from a large sample requires a balance of many parameters. Issues such as biofouling, membrane clogging, reduced viability of the target organism, low purity of the output sample, excessive processing time or low recovery may occur during the filtration process if the processing parameters and geometry are not chosen appropriately.
In single stage tangential flow filtration of blood culture where the target bacteria are to be retained, the membrane pore size is selected to prevent the bacteria (typically having a size of about 1 μm to about 2 um) from passing through the membrane pores. The pore size of the membrane is selected to be smaller than the typical size of the bacteria. However, if the membrane pore size is too small, it will result in a slow filtration process and potential retention of some undesired particles and debris in the retentate phase along with the bacteria. Membranes with very small pore sized require a larger trans-membrane pressure to drive the filtration since the flow resistance is typically higher as the pore size is reduced. If the membrane pore size is too large, the filtration time may be shorter but the target bacteria may pass through the membrane and the recovery rate will be reduced.
In tangential flow filtration, the tangential flow rate and trans-membrane pressure are normally chosen in order to provide a successful and efficient filtration. Desired efficiency or target efficiency means not only the percent recovery of the target bacteria and its purity are high, but also the filtration speed is optimized for the fixed filtration area. Higher tangential flow velocity generally helps prevent cake layer formation on the membrane surface or membrane clogging. The trans-membrane pressure is selected to provide a sufficient filtration rate but the trans-membrane pressure is not so large as to cause membrane clogging by pulling particulate matter into the membrane pores (reducing the effective diameter) or blocking the membrane pore entrances all together. This clogging occurs when the tangential flow velocity is too low even in devices having a low trans-membrane pressure. Membrane clogging can either significantly reduce the efficiency of the tangential filtration or make the filtration/purification process close to impossible. The membrane clogging problem is particularly severe when the solid content and/or viscosity of the sample is high as often seen in positive blood cultures.
In tangential flow filtration, the chamber geometry is an important factor which affects the filtration performance. There are typically two chambers in such devices, the chambers being separated by the membrane. The selection of chamber height, width and length affect the fluid flow resistance, which affects the tangential flow rate, velocity and effective trans-membrane pressure. For a fixed filtration area (width×length=constant), a higher chamber height will result in less flow resistance. In order to achieve enough flow velocity to prevent or minimize the cake layer formation on the membrane or membrane clogging, the flow rate has to be higher than the flow rate used for a low chamber height system. For most applications, a smaller chamber height provides for more efficient in filtration in each pass of the sample volume. The tangential flow resistance, however, is increased in this case, and a greater pressure differential is required to drive the flow along the membrane surface. Shorter chamber length and wider chamber width can reduce the flow resistance. Conversely, the introduction of an excessively wide uniform flow path at the sample inlet makes maintaining the flow velocity challenging. Hence, a proper corresponding geometry is required for the intended filtration parameters and performance for a specific input sample type and input volume.
In one embodiment, an example of the membrane device 600 can have a first chamber geometry of about 20 mm×about 42 mm×about 0.8 mm with a TEM membrane pore size ranging from about 0.2 μm to about 2.0 μm. More preferably, the TEM has a pore size in the range of about 0.4 μm to about 1 μm. Such pore size is selected to retain the bacteria in the retentate phase. Positive blood culture is introduced to a reservoir connected to the device inlet. The tangential flow is powered by a pump, syringe or pressure source etc. In this embodiment, the power source is a peristaltic pump which enables a recirculating flow allowing the sample to flow through the membrane device in multiple passes continuously in a setting similar to
The tangential flow rate can be in a range of 10 to 90 mL/min and more preferably in a range of 40 to 70 mL/min. The trans-membrane pressure can be in the range of 0.5 (about 3.45 kPa) to 10 psi (about 68.95 kPa) and more preferably in the range of 3 (20.68 kPa) to about 8 psi (55.16). The trans-membrane pressure is applied by using a pressure source, vacuum, a pump, a syringe, or other conventional mechanisms. Tangential flow drives the sample solution along the membrane, while a pressure gradient across the filter/membrane creates the motive force to drive smaller constituents or particles smaller than the pore size of the membrane through the membrane. The volume of the retentate phase decreases and the bacteria becomes concentrated as the filtration process continues.
To reduce the number of constituents in the sample, the sample can be pre-treated to remove some constituents to reduce the chance of membrane fouling. For example, when the sample is a positive blood culture, the blood cells in the background of the positive blood culture are removed prior to the sample filtration process. This can be achieved by prefiltration. In another example, the blood cells in the background of the positive blood culture are selectively lysed prior to the sample filtration process. The selective lysis of the blood cells can be achieved by using a liquid lysing buffer or a dried down lysing agent which is dissolved or mixed with the positive blood culture. It should be noted that careful selection of the differential lysing agent is critical so that the blood cells will be lysed efficiently and quickly without affecting the viability of the bacteria or their response to the drugs in AST test. Such gentle lysis buffers are well known to the skilled person and not described in detail herein. Dissolved salts, proteins etc. in the background of a positive blood culture can be removed by rinsing or washing with additional washing buffer such as deionized (DI) water. This process can be repeated as necessary to purify the retentate phase to satisfy the bacteria purity requirement for MALDI-TOF testing. The bacteria retentate may have additional lysing and washing buffers added to further reduce contaminants that may adversely affect identification and antibiotic susceptibility testing results. In some implementations, an antifoaming agent is added to the lysing buffer and/or washing buffer to prevent foam formation during the filtration process, as many positive cultures can create foams during the filtration process depending on the bacteria type. Foam formation during the process can be detrimental to the process and the quality of the output sample. The antifoam agent is selected and used in quantities that will not affect the bacteria viability, MALDI-TOF ID or susceptibility to drugs in AST test. Anti-foam agents are well known to the skilled person and not described in detail herein.
To test the system described herein, positive blood cultures were processed using a single stage tangential flow filtration with 8 different bacteria species in three types of BACTEC media. The bacteria tested were E. coli, S. aureus, S. pneumoniae, S. epidermidis, E. faecalis, K pneumoniae, P. aeruginosa and E. cloacae. Culture bottles used were a BACTEC standard bottle, a BACTEC plus bottle and a BACTEC lytic bottle. A blood sample (10 mL) was collected into the respective BACTEC bottle, which was then inoculated with a bacteria species to simulate a bacteria concentration of ˜50 CFU/mL in the blood volume. The sample was incubated in a BACTEC blood culture instrument until bacterial growth was detected. In BACTEC, growth is detected by monitoring changes in one of oxygen concentration, carbon dioxide concentration or a change in pH deemed indicative of microbial growth. The typical bacteria concentration in a positive culture ranges from about 106 CFU/mL to 108 CFU/mL. The positive blood culture (about 10 mL) is taken from the BACTEC bottle and lysed by mixing with 10 mL BACTEC lytic media (or other proper lysis buffer solution).
Traditionally, this mixing is performed manually by rotating by hand the container with the positive blood culture from an upright position to upside down position repeatedly. This mixing is sometimes also accomplished through additional features in the fluidic cartridge, such as a herring bone feature or bubbling the fluid. However, such a method is completely external to the fluidic path of the disposable cartridge. In one embodiment of the present invention, this mixing is automated and is accomplished by using a motor to rotate the disposable cartridge from an upright position to a rotation between 1-180°. Automation reduces hands on time for the technician, reducing labor costs.
The 20 mL lysed mixture solution is input to a tangential flow extraction system as described herein. A filtration device similar to membrane device 600 as described above was used. The tangential flow chamber geometry on the retentate side (i.e. the top side as illustrate) of the membrane is 20 mm in width×42 mm in length×0.08 mm in depth with a membrane pore size of 0.8 um. The tangential flow rate was 60 mL/min. The flow of the sample through the filtration device was powered by a peristaltic pump at the outlet side of the tangential flow filtration chamber. The output of the peristaltic pump was fed back to the sample reservoir attached to the inlet of the membrane device in an arrangement similar to that illustrated in
The above-described process can be modified to have more or less wash steps, more or less volume per wash, and more or less additional lysing steps depending on the requirements for processing time, sample purity, volumetric limitations, etc. Process parameters and/or geometry may be adjusted from those described above to suit a particular use of the method/device described herein.
(3) Bacteria Recovery and Concentration from Urine Sample
As previously mentioned, the membrane device 100 can be implemented for a single stage extraction such as where the sample tangentially flows along one membrane filter. Such a process may be implemented for bacteria recovery and concentration from urine in a process similar to that described in relation to the prior blood sample example. The following example illustrates how tangential flow extraction can be applied to a urine sample for bacteria extraction. The urine samples were acquired and all were culture negative prior to receipt. The same device configuration, as used for fresh whole blood TEM concentration experiments previously mentioned, with a pore size membrane of about 0.2 μm to about 3 μm was implemented for the urine processing. In the test, urine samples were spiked with E. coli at a concentration of 56 CFU/10 mL prior to the concentration process with a membrane device. A pressure control system (e.g. a system obtained from Fluigent) was used to provide 1 and 4 psi pressures on the input side (first chamber of the membrane device) to drive the multi-pass tangential flow. A 10 mL sample was thereby processed on a TEM device until the remaining volume reached 0.5 mL, which was recovered and plated on an EMB plate and cultured overnight. A total of five samples from separate subjects were used. In this preliminary test, the initial E coli concentration in the urine sample was 5.6 CFU/mL and the final E. coli concentration was 87 CFU/mL after the concentration process through the membrane device. The average recovery was 77% with a concentration factor of about 16-fold as shown in Table 2.
E. coli
E. coli
E. coli
E. coli
In another experiment, concentration of bacteria in urine sample showed a 10-fold increase in concentration of the bacteria from the original sample. β-lactamase (antibiotic resistance marker) was detected about 4 hours faster using TEM-implemented concentration of spiked urine samples than when using the unconcentrated urine specimen. Results are illustrated in the graph of
As previously mentioned, the membrane device 100 can be implemented for a multiple stage extraction such as where the sample components tangentially flow along several membranes such as where the membranes of each stage have different pore characteristics (e.g., size). Such a process may be implemented for bacteria recovery and concentration or even exosome extraction and concentration from fresh or diluted whole blood.
(1) Two Stage Exosomes Extraction and Concentration
Exosomes are extracellular vesicles that range in particle size from 120 nm to 30 nm found in blood or urine samples. They are secreted through the endosome multi-vesicular body complex by most cells and convey information to neighboring or remote cells by delivering RNA and proteins thus affecting various physiological and pathological signaling pathways. Exosomes have been widely investigated due to their availability through a non-invasive liquid biopsy process and their potential diagnostic value such as for cancer detection. Extraction of exosomes has been done by several techniques including the gold standard process using ultra centrifugation, precipitation and affinity-based or size exclusion column technologies. These technologies can be time consuming, tedious procedures or suffer from poor quality or low recovery efficiency.
The present technology permits a new process for extracting exosomes from biological samples such as using a multiple stage tangential flow extraction with membrane devices. Such a process is described with reference to
The second stage as illustrated in
Alternatively, additional stages can be added to the process in order to extract exosomes of different sizes and/or to increase the purity of the extracted exosomes. For example, after the first stage of plasma separation using 0.4 μm pore TEM and the second stage of 0.05 μm pore TEM process as described above, the sample (e.g., retentate from stage 2) can be further filtered using a membrane device with a membrane having a pore size on the order of 0.1 or 0.2 μm to remove larger particles. This third filtration stage can also be performed between the first stage and the second stage process.
In an example of the second stage filtration of
Particle size analysis using dynamic light scattering (DLS) showed that the hydrodynamic radius of the extracted particles matches the expected size range of exosomes. The particle size analysis as a function of trans-membrane pressure is illustrated in
(2) Two Stage Bacteria Extraction from Whole Blood, Diluted Blood or a Positive Blood Culture
When extracting bacteria from whole blood where a lot of red blood cells are present, a multiple stage process (at least two, or more) can be used to achieve the purity and concentration level of the target substance (e.g. bacteria). For example, a two-stage process can be implemented where the first stage process will remove large cellular particles (for example, red blood cells, white blood cells, or platelets) from the whole blood, diluted whole blood or whole blood culture sample and the larger debris (in case of lysed blood). The larger debris are particles greater than a predetermined pore size that will allow the bacteria or other target substance(s) to pass through the membrane. The bacteria remain in the permeate phase that passes through the membrane in the first stage process. An illustration of the tangential flow and trans-membrane (filter) flow allowing bacteria to pass through the membrane is illustrated in
In order to allow the bacteria to pass through the membrane device, a membrane with pore sizes on the order of the size of the bacteria or larger should be used. It was found that a track etched membrane with pore size on the order of about 2 to about 5 μm are suitable for this purpose. As described earlier, the membrane device includes a first chamber (an input chamber) and a second chamber (an output chamber) separated by a membrane (e.g., TEM). A trans-membrane pressure is applied to drive the bacteria through the membrane (i.e. into the permeate) while keeping the cells and platelets in the first chamber (i.e., the retentate). Both tangential flow and trans-membrane flow (as previously described) can be powered by pressure and/or vacuum sources at the input side and/or output side. The tangential flow can be one-directional flow. One directional flow can be a single-pass tangential flow or a multi-pass rotational flow in the first chamber such as a re-circulating flow powered by a fluid pump.
Alternatively, the tangential flow may be reciprocating flow where blood flow reverses its direction to allow fluid flow over the same membrane surface multiple times to drive the filtration process to completion at limited sample input volume and to reduce membrane fouling.
The second stage process may take the bacteria rich permeate from the first stage as the sample input and be processed using a secondary membrane such as a further TEM for tangential flow in another membrane device. This secondary TEM tangential flow process using a device having a first or upper chamber and a second or lower chamber separated by a TEM of pore size of about 0.2 μm to about 1.2 μm. As the bacteria rich permeate from the stage 1 filtration process is used as the input sample to second stage, and the sample flows along the track etched membrane in the upper chamber, liquid flows through the membrane by applying a trans-membrane pressure. The engineered system allows balanced dual flows along (tangential) and through (trans) the TEM membrane where bacteria are enriched, retained and remain viable in the first chamber. The tangential and trans flow rates are selected to prevent fouling of the membrane due to phenomena such as cake layer formation or pore clogging with particulate and to optimize the sample processing time without introducing undesired shear or lysing of cellular components. The bacteria remaining in the first or upper chamber can be recovered for next step testing such as detection, identification or antimicrobial susceptible test. Optionally, additional washing can be used to further purify the sample by removing dissolved salt, proteins, nucleic acids, peptide, lipids etc. such as using one or more additional membrane devices. The washing buffer or other suitable liquid can be introduced at the beginning, during or at the end of concentration process. The concentration process can be repeated after addition of buffer solution in order to reach a targeted concentration factor.
In an example of such a process, the performance of a larger pore size TEM device for a first stage in a multistage process was evaluated with bacteria in whole blood in BD BACTEC lytic (lysed blood) and BD BACTEC standard bottles (not lysed blood) although other types of blood culture media could be used. The initial screening used track etched membranes with pore sizes of about 2 μm and about 5 μm respectively. It was found that E. coli in the blood can pass through membranes with either a 2 μm and 5 μm pore size with high efficiency (>95%). In the graph of
A TEM device with a membrane pore size of 2 about μm was selected to conduct another test to understand the efficiency of separating blood cells from bacteria from the positive blood culture sample. A bacteria-rich blood sample was created by using E. coli spiked blood in a BACTEC Standard bottle with incubation. A single pass TEM device was used to separate the bacteria from the positive blood culture. A 5 mL bacteria-rich blood sample was processed by a single pass process powered by a vacuum source. Vacuum power was varied to study the effect of trans-membrane pressure on bacteria recovery and blood cell removal after the filtration process. The recovered bacterial solution was further analyzed to determine the residue complete blood count (CBC), plasma free hemoglobin and E. coli concentration. in order to estimate the purity of bacterial suspension. Results in Table 4 below showed that permeate yield, bacteria recovery rate, and residual CBC in the permeate phase do not change much as the trans-membrane pressure (TMP) changes. The only residue in the plasma/bacteria phase is a small portion of platelets. This is an indication that the process has a relatively large operating window. It should be noted that a multi-pass process was also conducted and it has similar separation performance as in a single pass process.
E Coli, %
From the above, it is clear that the permeate contains very little white blood cells and red blood cells, since the amount is below the limits of detection (LOD). The yield in the permeate was slightly higher for the devices with higher transmembrane pressure, with slightly higher recovery of E. Coli. This preliminary test results showed that the separation of bacteria from whole blood is possible.
As described above, tangential flow extraction can serve as a concentration and washing process that will rapidly isolate bacteria from positive blood cultures (either whole blood samples or diluted blood samples) for identification and/or antibiotic susceptibility testing. In an example of such a process, about 8 mL to about 10 mL of patient blood sample is collected into a BACTEC bottle, and inoculated with bacteria such as E. coli, S. aureus, S. pneumoniae, and other bacteria. The sample is incubated in a BACTEC blood culture instrument until a positive blood culture is detected. Within about 8 hours or less from detection, the positive blood culture is applied to a tangential flow extraction process where bacteria will be isolated and washed. The membrane device 600 (or tangential flow extraction device), similar to the previously discussed examples, has a first chamber 610 (e.g., input or upper chamber) and a second chamber 620 (e.g., output or lower chamber) separated by a track etched membrane (TEM) 630. The sample flows (repeatedly if a multi-pass process is being used) along the track etched membrane in membrane device 600 in the first chamber (e.g., upper chamber (104 in
In another example, the tangential flow membrane device technology may be implemented to provide a bandpass filter solution(s) employing a two-stage process that separates/extracts constituents of interest, such as pathogens of interest, from both larger and smaller components in the sample based on inherent size differences of the various components/constituents. The filter passband, such as the one illustrated in
1. High-pass Filter membrane device. One stage of filtration employs a membrane device as a high-pass filter that passes smaller components of a sample, such as a bacterium of interest, while excluding larger components, such as red and white blood cells. The high-pass filter membrane device employs a membrane (e.g., TEM) with pore size(s) in a range sufficiently larger than about 0.5 μm and less than about 8 μm such as in a range from about 2 μm to about 5 μm in diameter to establish a cutoff size that excludes blood components that are typically larger than about 5 μm in diameter.
2. Low-pass Filter membrane device. In another stage of filtration, a membrane device is employed as a low pass filter. For example, the bacteria and accompanying debris that passed through the high-pass filter membrane device is then subsequently processed with a membrane device serving as a low-pass filter. The low-pass filter uses a membrane such as a TEM with chosen diameter pore size(s) to retain the relatively large bacteria and allow debris, analytes and waste to be removed. For example, such a pore size may be on the order of about 0.4 μm to about 1 μm for the low-pass filter membrane.
The end result is the sample of interest separated from the blood background, in a rapid fashion (e.g., on the order of about 5 to about 20 minutes), and ready for downstream processes, such as identification testing, resistance marker testing, susceptibility testing, plating, measuring turbidity, culturing, sequencing, and/or other processes that are routinely performed in a microbiology, clinical, or research laboratory.
Such an example process is further illustrated in the process flow chart of
Such technological processes as described in this specification may have various benefits. For example, the technology could facilitate identification and antimicrobial susceptibility testing directly from a positive blood culture (PBC) without the need for further subcultures, reducing time to results by as much as approximately eighteen (18) hours or more. This reduced time to results can have significant positive impact on clinical therapeutic decisions, resulting in improved patient outcomes, as well as institutional cost savings.
Current methods for processing a PBC include traditional subculturing or centrifugation techniques. Previous attempts are severely limited by processing times and complicated procedures.
Alternatively, traditional centrifugation techniques may be employed to isolate bacteria directly from the PBC to overcome this delay, but they require a number of laborious centrifugation and washing steps to obtain a pure sample ready for downstream ID/AST tests.
The proposed technology may overcome the limitations of such processes by providing a solution that isolates the sample of interest from the PBC in a short period of time, such as less than about 15 to 30 minutes, without the need for multiple centrifugation or washing steps, both decreasing the time to results and the number of laborious procedures. Further, the present technology can be readily automated, unlike centrifugation approaches, to enable high throughput processing.
Previous technological attempts to directly process a PBC using filtration traditionally relied on dead-end filtration techniques. In dead-end filtration, the feed solution is only driven perpendicular to the membrane and the resulting pressure is used to force particles smaller than the pore diameter across the filter. The particles larger than the pore diameter, however, build up on the surface of the membrane to form a cake that eventually fouls the filter. Common solutions to overcome this limitation is to lyse and digest the PBC sample prior to filtration in an attempt to reduce the overall size of particles to restrict the cake from forming. Previous studies have shown though that lysing and digesting the sample has a negative impact on the viability of the target microorganisms that impacts the performance of downstream ID/AST studies, effectively precluding prior art attempts from adoption.
The proposed technology as described herein can rely on tangential flow filtration that offers significant advantages over dead-end filtration such as to prevent filtration clogging. Tangential flow filtration (as described in detail herein) continuously washes the filter as the solution is fed tangentially along the membrane to prevent fouling, such as with a peristaltic pump in a multi-pass operation. It also can provide a benefit of repeated filtration of a given sample so as to improve concentration while avoiding clogging. As a result, the PBC input (as is, lysed or digested) can be more flexible for this tangential flow filtration, further reducing complexity and increasing performance.
As discussed herein, the apparatus with bandpass filter membrane devices may be enabled with tangential flow filtration (a crossflow filtration). As described, tangential flow filtration (TFF) may be understood to be a type of filtration that passes the feed solution (the sample) tangentially along the surface of the membrane rather than merely directly perpendicular as in dead-end filtration. A significant advantage of TFF is that the tangentially flowing feed solution reduces caking the membrane and fouling the membrane pores, effectively increasing both performance and duration of operation. Repeated flow of the solution over the same surface area reduces the opportunity for loss of targeted microorganisms to the membrane surface, since effective filtration can be achieved by multiple passes of the sample over the same surface. Additionally, since the sample is exposed preferentially to the membrane top surface as opposed to a large interior surface area (as with fibrous membranes), there is reduced opportunity for loss of target microorganisms due to adherence to the membrane compared to approaches where the sample is allowed to pass through a thickness of fibrous or another higher interior surface filter. Additionally, a pressure differential is formed normal to the membrane that serves as a motive force to drive particles smaller than the diameter of the pores across the membrane, such as by application of a negative pressure differential (e.g., a constant negative pressure differential) held with a vacuum or vacuum pump.
The examples of the disclosed technology can isolate bacteria of interest from the PBC (or other biological sample) by separating sample components based on inherent differences in size using, for example, a bandpass approach. The technology can employ multi-pass tangential flow filtration as described herein.
Optionally, a disposable cartridge is used to isolate bacteria from the other parts of the sample using a tangential flow membrane device. The operation of one example of such a disposable cartridge is described in
The sample is drawn through a filter screen or porous membrane 815. The pore size of the filter or porous membrane is selected to prevent resin in the syringe or culture bottle from flowing into main chamber 820. The pore size for a filter or membrane that will selectively prevent the resin from passing through the membrane or filter, yet allow other sample constituents to pass through, is well known to one skilled in the art. Membrane pore sizes in the range of about 25 μm to about 100 μm are contemplated, but the skilled person may select another pore size if so warranted by the resin particle size. Resin particles are typically at least about 10 μm or more in diameter, which is much larger than the other sample constituents (red blood cells, white blood cells, microbial contamination (if present) etc.
Although illustrated as a seemingly planar arrangement, in one embodiment the port 810, the filter 815 and the vial 845 are on one side of a cartridge substrate (not shown) and the main compartment 820, filter 835, wash chamber 830 and other components for process the sample and illustrated in
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The cartridge described herein solves these issues and facilitates an advantageous work flow. In the first step, the PBC bottle is attached to a cartridge. This is illustrated with reference to
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Currently, the vials 1730 that are commercially available contain a screw cap and require manual sterilization of the cap before attaching to the disposable cartridge 1720, and before the user accesses the vial 1730 with a syringe. Current bottles 1710 would require a secondary method for attaching the vial 1730 to the disposable cartridge 1720 to maintain positioning during processing. In some embodiments of the present invention, the vial 1730 securely connects to the cartridge 1720, thus preventing user exposure, and allowing the user to access the contents of the vial 1730 once under a biosafety cabinet via a syringe or pipette. In one embodiment of the present invention, the cap of the vial 1730 has several features, including the following illustrative features, which may be present independently of one another: (1) The cap can contain a sponge or other material that contains a disinfectant. This ensures any small amount of liquid that escapes during detachment is trapped in the sponge and sterilized. (2) The cap can also contain a built-in septum, such as a rubber septum, that a needle can easily pierce and will reseal after removal. The septum facilitates the release of the vial 1730 from the cartridge 1720 without risk of spilling or leaking. (3) The cap can also be threaded such that it can be unscrewed from the vial 1730 once in a biosafety cabinet. This gives the user several options for accessing the highly concentrated microbial sample, with safety measure to reduce the risk of the user. (4) The cap can contain a mechanical notch that locks the output vial 1730 into the disposable cartridge 1720 to ensure the vial 1730 does not dislodge from the cartridge 1720 during processing. (5) The system 1700 may pull a vacuum on the output vial 1730. This vial 1730 can then drive fluid flow into the vial 1730 as a final step of the processing.
Although the technology herein has been described with reference to particular examples, it is to be understood that these examples are merely illustrative of the principles and applications of the technology. In some instances, the terminology and symbols may imply specific details that are not required to practice the technology. For example, although the terms “first” and “second” may be used, unless otherwise specified, they are not intended to require any order but may be utilized to distinguish between distinct elements. Furthermore, although process steps in the methodologies may be described or illustrated in an order, such an ordering is not required. Those skilled in the art will recognize that such ordering may be modified and/or aspects thereof may be conducted concurrently or even synchronously. In this disclosure, when a range is provided it is understood that the range may include any value within the range as well as the limits. Approximate values may be utilized and will be understood to include all values significantly near a stated value with reference to the stated value's significant digits.
It is therefore to be understood that numerous modifications may be made to the illustrative examples and that other arrangements may be devised without departing from the spirit and scope of the technology.
This application claims priority from U.S. Provisional Application Ser. No. 62/796,757 filed Jan. 25, 2019, which is incorporated by reference herein. This application also claim priority from U.S. Provisional Application Ser. No. 62/906,822 filed Sep. 27, 2019 which is also incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/014758 | 1/23/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62906822 | Sep 2019 | US | |
| 62796757 | Jan 2019 | US |
