Microfluidic Technology Holds The Future Of Blood Cell Separation

Compared to other conventional blood cell separating tools like automated FACS and MACS, microfluidic cell sorting offer faster and higher output, simpler operating procedures, reduced costs, and improved purity of sorted samples.

Apheresis has been at the forefront of therapeutic approaches for an increasing number of indications caused by the formation of pathologic antibodies treated by therapeutic plasma exchange, or through the use of red cell exchanges to overcome complications secondary to sickle cell crises. Likewise, the number of hematopoietic stem cell transplants has continued to grow annually and this is the direct result of the expansion of apheresis collections. Millions of blood components including red blood cells, platelets, and granulocytes are being transfused each year in the country. These different hematologic components perform distinct functions in vivo and thus the ability to efficiently fractionate blood into its individual components has innumerable applications in both clinical diagnosis and biological research. Yet, processing blood is not trivial. While there are numerous methods for separating blood components, a vast majority utilize centrifugation-based processing. Although widely used, these cell separators have limitations (such as hemolysis) that may outweigh their advantages in some settings. There has, therefore, been a significant research effort in recent years for the development of novel centrifugation-free approaches for blood cell separation. Some of these emerging technologies are already transforming niche applications, poised to enter mainstream blood cell processing in the not too distant future.

Over the years a number of apheresis platforms have been utilized. Conventional plasma and leukocyte separation methods have relied on membrane-based filtration. However, the high cellular fractions lead to membrane clogging and compromise separation efficiency. In biomedical research and clinical diagnostics, along with filtration, centrifugation, and sedimentation techniques, fluorescent activated cell sorting (FACS) and magnetically activated cell sorting (MACS) have become standard methodologies for accurate and continuous separation of blood cells. FACS is capital intensive and requires substantial attention from highly trained staff. As a result, FACS is challenging to scale for large clinical studies with many samples. While MACS is most often practiced manually or on proprietary instrumentation with a capacity to run batches of 6 to 10 enrichments. Although the potential to automate MACS at higher throughput using pipetting robots exists, this also requires large samples for good results, would be capital intensive, requires extensive custom programming, and is only effective for central processing centers with a large steady supply of samples. Both FACS and MACS technologies have thus reached maturity, so their improvement to achieve lower cost, higher portability, smaller sample sizes, and greater purity has become a difficult task. 

These factors have led many researchers to study alternative methods of cell separation such as acoustic separation, dielectrophoresis, hydrodynamic separation, and microfluidics. Enormous progress has been made in the past few years in the area of microfluidics-based blood component separations. Microfluidics leverages its many distinct advantages such as low sample volume for processing blood, as these samples are precious and rare especially in the case of neonatal care. Moreover, computer innovations have made it possible with current apheresis technology to obtain more real-time information of the procedure which permits the operator to adjust parameters not only to optimize the specific procedure but also to safeguard against potential adverse events.

Indian scenario
With several road accidents occurring every day, millions of surgeries and major operations being performed every year, rising cancer-related procedures like chemotherapy, and increasing pregnancy complications, all require a serious call for transfusion of blood and blood products. Thus, a well-organized blood transfusion service (BTS) is a vital component of any healthcare delivery system and ensuring the safety and availability of blood and blood products is an essential public health responsibility. The BTS in India is highly decentralized and lacks many vital resources like manpower, adequate infrastructure, and financial base. The blood component production/availability and utilization is extremely limited. For quality, safety, and efficacy of blood and blood products, well-equipped blood centers with adequate infrastructure and trained manpower is an essential requirement. A major problem plaguing blood banks in India is poor monitoring and control because of the multiplicity of agencies involved. Consistent quality and safety in the provision and administration of blood and blood products cannot be achieved without a coordinated service with an appropriate national blood policy.

The Government of India has adopted the National Blood Policy in 2002 which aims to develop a nationwide system to ensure easy access to adequate, safe and good quality blood supply and its components. National Blood Transfusion Council (NBTC), the apex policy-making body for issues pertaining to blood and plasma is a part of the National AIDS Control Organization (NACO). NACO supports a network of over 1100 blood banks across the country and strives to achieve accessibility to an adequate quantity of safe, quality, and affordable blood and blood components to the needy. NACO through different phases of National AIDS Control Program (NACP) supports the establishment of component separation facilities and also funds modernization of all major government and charitable blood banks at state and district levels. In order to promote rational use of blood, Blood Component Separation Units (BCSU) are being established and NACO support is extended to several BCSU across the country. These BCSUs are working in their respective states and the proportion of blood units processed for component separation has risen since the launch of NACP.

New principles of safe handling, cost, and administration of blood components have an impact on blood safety with the growing concern for apt utilization of blood products. Without blood or blood products transfusion, effective management of severe trauma, major elective surgery, and serious obstetric complications is not possible, as it is an essential part of the infrastructure. NACO is working hard to improve BTS in the country, but there is still no national blood law. In the absence of a legal authority empowered by the national blood law, optimal quality of BTS will remain a mirage in the country.

Global market
The apheresis market was valued at USD 1983 million in 2018 and is expected to register a CAGR of about 10.89 percent from 2018-2023, according to Mordor Intelligence. Apheresis has recently witnessed a great demand due to an increased number of patients suffering from various ailments related to blood, kidney, metabolic diseases, and neurological disorders. It has been observed that apheresis technology has been used in order to reduce the number of white cells until they can be controlled by other medications during cancer. According to the Leukemia & Lymphoma Society (LLS), approximately every three minutes, one person in the United States is diagnosed with blood cancer. An estimated combined total of 174,250 people in the United States are expected to be diagnosed with leukemia, lymphoma, or myeloma in 2018. According to the Cancer Research UK, it is estimated that the incidence rates for leukemia are projected to rise by about 5 percent in the UK between 2014 and 2035, to 19 cases per 100,000 people by 2035. Along with these main disease conditions, the technology has also been widely used for the treatment of conditions, such as myasthenia gravis, systemic lupus disease, severe rheumatoid arthritis, and in cases of selected organ transplantation with a high risk of antibody-mediated rejection of the transplant. Use of apheresis in clinically ill patients is increasing day by day. It has been widely used as a primary therapy or as an adjunct to other therapies for various diseases, such as thrombotic thrombocytopenic purpura, haemolytic uremic syndrome, drug toxicities, autoimmune disease, sepsis, and fulminant hepatic failure. With the steady increase in the number of patients suffering from these diseases, ailments, and the technical ease of using these devices in the treatment of these diseases, requiring a minimal hospital stay, the global apheresis market is poised to grow.

The global market is majorly driven by the rise in the use of apheresis equipment during blood donation. Moreover, the procedures that are accomplished by apheresis equipment are also used as the second line of treatment in many other conditions. Increasing insurance coverage, a growing number of new product launches, and rising healthcare spending is another factor helping the revenue growth of the apheresis equipment market. However, high costs associated with this equipment along with strict regulations are anticipated to hinder the growth of the market. The disposables have the largest share in the market. The factor which augments the growth of the disposables in the market are bulk purchases which are one of the important factors. Also, manufacturers establish long-term purchase contracts with the end users in order to achieve bulk purchase benefits and strengthen their customer relationships.

North America is expected to have the largest market share owing to the highly sophisticated healthcare infrastructure and high healthcare expenditure. In addition, the existence of a high number of market players, high donor awareness, the demand for plasma-derived medicines, rise in the disposable income, and relatively higher number of research and development exercises relating to product manufacturing and marketing accomplishments make this region dominant in the market. Asia-Pacific is also expected to grow at a high rate due to a considerably well-established apheresis market in Japan, moreover, the emerging economies, such as India and China, have large patients pool, geriatric population, and rising healthcare expenditure. Growing occurrence of diverse types of hematological disorders will also boost the market growth in the Asia-Pacific region.

Major firms are focusing on small firms with a working methodology of securing the end goal to manage the position in the market and are associated with mergers and acquisitions, key joint efforts, and novel item advancement to pick up profit share in the business. In 2018, MedAware Systems, Inc launched a comprehensive database on apheresis through its SOHInfo division, carrying data from clinical trials, cohort studies, medical journals and others. Some major key players in the global market include Haemonetics Corporation, Fresenius Kabi, Asahi Kasei Kuraray Medical, Cerus Corporation, Terumo Corporation, HemaCare Corporation, Kaneka Corporation, Kawasumi Laboratories, Therakos, Mallinckrodt, and B. Braun Melsungen among others.

Technological advances
The new technologies in cell separation have evolved significantly from the cumbersome and crude age-old technologies. There are several centrifuge-free options of blood cell separators presently available in the market.

Membrane filtration. Membrane filtration technology present an attractive alternative to the centrifugation-based cell separation since filtration systems are significantly simpler and less expensive. Recent developments in microfiltration utilize membranes for the absorption of unwanted solutes and cells present in the blood. For example, HemoSep, a newly developed hemoconcentration system, soaks up to 250 mL of excess plasma during blood salvage without the use of centrifugation. The primary component of the HemoSep system is a blood bag separated into two compartments by a filter membrane, which prevents cells in the blood compartment of the bag from entering the super-absorber pad contained in the other compartment of the bag. The blood plasma and other fluids are free to pass through the membrane, however, to be soaked up by the super-absorber pad. The low-cost, simplicity, and portability of the HemoSep system in comparison to conventional autotransfusion devices makes it an attractive alternative for blood salvage, particularly in resource-limited settings. Filtration using spinning membrane is another method used for blood cell fractionation. Spinning membrane separators provide excellent filtration rates by generating Taylor vortices in the gap between the membrane and the shell of the device. This unique flow pattern creates flow at the membrane, constantly sweeping the surface to prevent the cellular components from depositing on and clogging or fouling the membrane, while continually replenishing the medium to be filtered.

Microfluidics. Microfluidics technology has been adapted in clinical and biomedical applications, where it is frequently known as the lab-on-a-chip (LOC) technique. Compared to other conventional cell separating tools like automated FACS and MACS, microfluidic cell sorting offer faster sorting rates and a higher output; simpler operating procedures, portability, and reduced costs, reduced biohazard risks, and improved purity of sorted samples. The ultra-small dimensions of microfluidic technologies make it easier to manipulate cells and enable faster detection which makes them perfect for in-situ testing. Microfluidic or LOC devices are capable of integrating as well as miniaturizing multiple laboratory procedures which are  highly desirable in the ever-advancing biomedical and biotechnological translational research. Microfluidic sorting has great potential in isolation highly pure cells for use in biomedical and clinical applications. At present, microfluidic devices integrated with FACS are used in isolating stem cells, lymphocytes, and circulating tumor cells (CTCs).

Dielectrophoresis-based cell separation. Dielectrophoresis (DEP) has been demonstrated as an effective mechanism for cell sorting in microfluidic settings. Many existing methods utilize sophisticated microfluidic designs that require complicated fabrication process and operations. Using a simple array of indium-tin oxide (ITO) electrodes to generate DEP force field, the cells of interest are hydrodynamically separated from the blood. It causes cell separation to be done 10,000 times faster. Improving the ability to separate particles and cells in a continuous flow pattern facilitates faster and incessant medical diagnosis. Thanks to the new design of these cell separators, they can be used in vital therapies for life-threatening diseases, can be carried out at a fraction of the usual cost, and can be used for therapeutic as well as research purposes.

Optical sorting. Optical manipulation and separation of cells use a focused laser beam to trap cells owing to the difference between the refractive indices of the cell and its surrounding fluid. Optical forces can be applied from the outside of the microfluidic device, thus allowing development of highly modular, multi-purpose systems for cell sorting and manipulation. Optical forces offer more interaction freedom, which can be adjusted in real-time. Optical sorting principles have been used to adapt FACS on a miniaturized scale. Microfluidic optical tweezer devices have also been designed for high precision single-cell isolation. Optical cell sorting microsystems have also been devised that utilize more than one wavelength of laser beam to sort several populations. The different optical tweezers are able to separate the cells based on the cell size, fluorescent intensity (inherent or from labeled antibodies), and the laser power.

Acoustic separation. Acoustic cell separation device can manipulate thousands of cells, separating the tumor cells from the blood cells or specific blood cells from whole blood, based on their significant difference in size, compressibility, and other physical properties, by exposing them to sound waves while flowing through the microchannel. It offers a unique approach for researchers in bioengineering projects and clinical diagnosis. Separating the cells with sound offers a gentler alternative to the existing cell sorting techniques, which requires the labeling cells with antibodies or exposing them to stronger mechanical forces that may damage the cells. While there are some white blood cells that have overlapping acoustic properties with cancer cells, a platform, which utilizes the negative acoustic contrast elastomeric particles with CD45-binding whole blood cell (WBCs), is developed to further reduce the WBCs’ background. This platform uses an integration of three modules of microfluidic platforms, which consist of a high-throughput separation, cell spatial organization and cell staining, imaging, and quantification analysis. This platform combines the isolation and evaluation steps toward rare cancer cell sorting and identification.

Intelligent image-activated cell separation. A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. A new machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell separation at an unprecedented rate is now being developed. This technology, which is referred to as intelligent image-activated cell separation, integrates high-throughput cell microscopy, focusing, and separation on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. The technology can be used for real-time sorting of particular blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Road ahead
There has been the development of several microfluidic approaches that exploit the different physical characteristics of cells, fluid mechanics, and biorheology of blood as well as the difference in binding affinity of specifically targeted cells to separate and/or enrich them. The manufacturers will continue to push the boundary for developing centrifugation-free blood cell separators as more creative and innovative technologies continue to be developed and introduced in the near future. It is likely that they will cater not only for the processing of both large and small volumes of blood, but are also fast and efficient and with high sensitivity, purity, and yield. However, for these blood-based separation systems to be realized as true point-of-care devices, these technologies and devices must be simple, easy to use, and with little or no sample preparation required. This will set the challenges for developers and will by no means be easy, but possible. While these centrifugation-free technologies are very promising, they typically serve niche applications and many are still in early stages of development. Further research is needed to move these emerging technologies into the mainstream of blood cell separation, to provide the highest quality blood products for transfusion and research, and ultimately improve clinical outcomes for the patients. Overall, it is envisioned that the microfluidic devices can become a low-cost, rapid, and multi-functional tool for blood cell separation in resource-limited environments or point-of-care settings.