Diagnosing Similar Diseases in Low Resource Settings

Diagnostics in Low-Resource Settings

A lot of excitement surrounding microfluidics has been about its promising use in diagnosis in low-resource settings. Many infectious diseases present in developing countries are manageable or treatable with available medications, but still account for 1/3 of deaths. In these areas, multiple diseases present similar symptoms, leading to misdiagnosis and thus incorrect treatment. Hundreds of blood-based microfluidic immunoassays are available for diagnostic purposes, but they’re not all created equally. They require varying levels of sample processing or analysis that prohibit their deployment in low-resource settings. Further, while some diseases may have similar symptoms, they might require different detection techniques, with varying sample volumes, reagents and processing time, making it difficult to detect multiple diseases within the same system. This is the focus of recent work from Paul Yager of University of Washington. In his Lab on a Chip paper, “Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards,” he and his colleagues develop a system to detect both Typhoid fever and malaria.

Malaria and Typhoid Dual Detection

The proposed card is compact and intended to integrate with the DxBox. All sample processing occurs on-card including IgG filtration. This also depicts the porous nitrocellulose membrane of the FMIA which provides high assay surface area.

The proposed card is compact and intended to integrate with the DxBox. All sample processing occurs on-card including IgG filtration. This also depicts the porous nitrocellulose membrane of the FMIA which provides high assay surface area.

The developed system is intended to integrate with the DxBox, an ongoing project focused on a point-of-care diagnostic device. As I mentioned before, different diseases might require different means of detection. In this case, the researchers decided to detect antigens generated by malaria parasites and IgM antibodies generated by the host in response to the bacteria responsible for typhoid (Salmonella Typhi). The microfluidic card is based on a flow-through membrane immunoassay (FMIA) composed primarily of nitrocellulose, instead of traditional microfluidic channels. Nitrocellulose is essentially paper and provides a lot of surface area, creating shorter assay times. Enzyme-linked immunosorbent assays (ELISA) are standard lab assays and will be replicated using FMIA. However, ELISA can be slow (more than 3 hours) due to the diffusion between the bulk fluid and the capture service, while the FMIA can perform the same task in half an hour due to its high surface area.

Immunoassay Process

The system processes blood from the same sample in different, parallel steps to test for malaria and typhoid.

The system processes blood from the same sample in different, parallel steps to test for malaria and typhoid.

The detections of both analytes are run in parallel and start with the same unfiltered blood. The card extracts the plasma with a filter, eliminating whole blood cells, and this is where the assays for malaria and typhoid diverge. The typhoid assay must filter out any IgG antibodies (which would cause false positives when testing for IgM) and dilute the sample further. This results in a four-fold increase in the sample volume used in the malaria segment. Each analyte is then captured by immobilized reagents and labeled with gold nanoparticles conjugated to antibodies. The entire process is driven by pneumatic pressure and valves. Pneumatics is cheaper than alternatives, plus it doesn’t dilute the sample with an additional liquid, but it comes at the cost of introduced air bubbles. Air vents were incorporated to eliminate bubbles, but they were not totally eradicated and still obstructed the image analysis sometimes. Within the DxBox, analysis is intended to be carried out by a webcam. However, the current design of the system created nonuniform lighting (which can be rectified), and a flatbed scanner was used instead.

Results

This microfluidic card was tested on blood samples with Typhoid or malaria. Unfortunately the researchers did not test on a large enough sample to evaluate clinical utility or determine a limit of detection for the card. Currently lab-based ELISA has a limit of detection near 4 ng/mL, which is clinically relevant. The researchers also ran each sample on ELISA and a bench-top FMIA in addition to the on-card FMIA. Comparing the quantified signal of the on-card FMIA to ELISA resulted in an R2 value of 0.73, and on-card FMIA vs bench-top FMIA had an R2 value of 0.92. These are fine results that demonstrate how closely the on-card FMIA follows the bench-top methods, but it would mean a whole lot more given a limit of detection.

Discussion

The results of this card design seem promising but will mean a lot more with more testing. The pneumatic actuation was a major hindrance to project success. While they could operate at different pressures, the actuators were unable to actually control the liquid velocity. Also, the pneumatics introduced bubbles into the card, which not only affected the assay process but the final image to be analyzed as well. While only two diseases were showcased here, the authors have indicated that there is already work to create a more complete fever symptom panel. They also acknowledged that this format could be applied to other panels aimed at diarrheal diseases and sexually transmitted diseases as well. This format could really be adapted for a variety of diseases, with the disease diagnosis as the limiting factor for card design. 

Reference:

ResearchBlogging.org

Lafleur, L., Stevens, D., McKenzie, K., Ramachandran, S., Spicar-Mihalic, P., Singhal, M., Arjyal, A., Osborn, J., Kauffman, P., Yager, P., & Lutz, B. (2012). Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards Lab on a Chip, 12 (6) DOI: 10.1039/C2LC20751F

Microfluidics? What's That? A Beginner's Guide

Author's note: This post was chosen as an Editor's Selection at ResearchBlogging.org. Thanks for the support!

 

Microfluidic chemostat used to study microbes

Microfluidic chemostat used to study microbes

I don’t quite have the resources to poll the United States and the rest of the world, but if I did, this is what I’d ask:

  1. Do you know what microfluidics is?
  2. Can you explain it to me?
  3. Do you currently use anything with this technology?

We may never know the results of the poll, but I think I'd hear "No" and "What is microfluidics?" Have no fear, because today you’re lucky enough to read my Beginner’s Guide to Microfluidics.

To start with, let’s look at the word itself. It is the word fluidics with the prefix micro- strapped onto the front. Thus, we’re looking at the manipulation of fluid at the micro scale, often flowing through channels. There isn't a strict definition, but some like to refer to flow with a channel dimension less than 1,000 µm as microfluidics. It's more complicated than that, so to guide us, I’ll be referencing heavily from a review article George M Whitesides wrote in 2006, “The origins and the future of microfluidics.” For those unfamiliar with Dr. Whitesides, he is a legend in many spheres including microfluidics, and if you are reading a paper on microfluidics, it most likely either cites him, or can be traced to something that does. In this particular article, he looks at four “parents” of microfluidics that have brought us to the technology we have today:

  1. Microanalytical methods
  2. Biodefense
  3. Molecular biology
  4. Microelectronics

Microfluidics’ Loving Parents

Microanalysis

Microanalysis is the chemical analysis and identification of small amounts of matter, such as capillary electrophoresis. First developed in the 1960s, it is a method to separate ionic species using their size to charge ratio. Capillary electrophoresis is still used today, showing that microanalysis not only provided needs for microfluidics to meet, but it also provided some techniques.

Biodefense

Awareness of chemical and biological warfare escalated after the Cold War, fueling the United States’ Defense Advanced Research Projects Agency (DARPA) to heavily fund microfluidics research in the 1990s. Biodefense remains a major area or research in homeland defense as demonstrated by the recently unveiled microfluidics-based device that detects anthrax or other suspect DNA. The suitcase-sized system recovers cells from a sample and analyzes the DNA in less than an hour, while it currently takes 1 to 3 days in the lab. 

Molecular Biology

In the past few decades, genomics activity has progressed quickly and has demanded newer technology to grow even further. Microfluidics had the opportunity to process DNA faster, cheaper and more precisely. Stephen Quake, a professor at Stanford University, has been leading cutting-edge research in sequencing. The ability to sequence DNA in weeks rather than months has allowed him to start a company, Helicos, implementing microfluidic methods. There have been several microfluidic applications for sequencing as researchers and companies race to sequence a human genome for only $1,000.

Microelectronics

While the three previous “parents” have provided some tools and needs for microfluidics, the final “parent” helped it find its form and substance. Microelectronics was also developing at the time (although it was further along), and people saw a way to manufacture micro-sized structures precisely. Microfluidic components were first made of silicon and glass with microelectronics techniques such as photolithography. Eventually, silicon was replaced by materials with more suitable properties. Silicon is not durable, cheap nor transparent to visible or ultraviolet light. This is important because light is often used to either determine the results of a test, or to or to provide excitation energy. The most popular substitute material has been PDMS (polydimethylsiloxane). PDMS is a soft, transparent elastic polymer that is permeable to carbon dioxide and oxygen, making it useful for housing cells. Other cheaper, alternative materials have also been developed, such as thermoplastics, paper and string.

Advantages of Microfluidics

These four parents have given birth to a field that focuses on biochemical and medical applications, and with good reason. While everyone loves small things (I’m sure that micro is sad that it never got its time in the sun before the world became obsessed with nano), there’s more to it than that. A microfluidic device does have a smaller footprint than a huge machine in a lab, but it also uses a lot less fluid too. Why would you make a whole pot of soup just to taste and then dump it down the drain? On the other hand, I would make as little as I could and still taste it. So we can see that microfluidics is beneficial because it uses less costly chemicals and reagents, but it's also attractive due to the physics happening at the micro scale. Now, we’re not talking about leaving the world of classical physics and entering quantum physics, but matter does behave differently, most evidently in the way liquids flow.

Turbulent flow vs Laminar flow

Turbulent flow vs Laminar flow

Laminar flow around air bubbles ( source )

Laminar flow around air bubbles (source)

In fluid mechanics, there are two main types of flow: laminar and turbulent. Turbulent flow is just how it sounds, violent and chaotic. Laminar flow is peaceful, and particles will basically maintain their course without mixing. The state of a flowing liquid is determined by its Reynolds number. This is proportional to the dimensions of the channel and the velocity of the liquid. Slower liquids travelling in narrower channels are more likely to be laminar. This not only makes pretty images like those from Albert Folch’s lab, it gives a new level of control to researchers. Two laminar fluids flowing side-by-side will only slowly mix by diffusion, rather than convection. This allows us to do some clever manipulations, like selectively inserting components to induce mixing in some of the streams in a multi-stream channel. Other techniques exist to create droplets, which allow for controlled mixing, and capillary action is utilized to eliminate the need for pumps.

Microfluidic Applications and Issues

Paul Yager  with a lab-on-a-chip that  detects malaria

Paul Yager with a lab-on-a-chip that detects malaria

Microfluidics can be incorporated into a lab-on-a-chip which is used as an assay or diagnostic test and replaces its large, costly full-size lab counterpart. This has three basic components, a sample, a process and a validation method. For example, you might input a fluid such as saliva or blood to be analyzed. Next, you’ll have to process this blood mechanically or chemically. This may mean mixing it or applying filters to isolate what you want, and then introducing a possible chemical reaction. Even so, what good is this reaction if you can’t tell if it happened? You may be able to see a change in color, or look at it under a fluorescent microscope. These three components can be fulfilled in dozens of ways to create whichever test you so desire.

You must be saying to yourself, “Wow! What a technology! So why haven’t I heard more about this if it can do so many magical things?” Well, space travel is cool too, but when was the last time that you were out in space? Microfluidics just hasn’t made its breakthrough to the rest of us consumers. We can create some pretty cool applications with it, but how often can those prototypes be easily mass-produced? And how easy will they be able to use and troubleshoot and fix? Both concerns are even more important for point-of-care applications. Point-of-care refers to a medical application that is being used right where the caregiver meets the patient. In the developed world, this means that your samples don’t have to be sent to a lab somewhere to be processed, and in the developing world, this gives access to life-saving diagnostics that are normally too expensive to purchase and operate. More importantly, the quick response time of the devices would provide results to people who have travelled to a far-away clinic and need to return home again. No matter how elegant or complex a microfluidic device is, it must be easy and cheap to make, simple to use and durable.

Now, I don’t want it to seem like there are no companies producing microfluidics-based technologies. Some relevant ones are Helicos, FluidigmClaros DiagnosticsMicroFluidic Systems, Affymetrix and Advanced Liquid Logic. If you want to see a more complete list, check out the one at FluidicMems. I hope that I’ve helped you to understand the basics of microfluidics, and you can continue to learn about the field on this site. Oh, and if you were wondering how long it will be until you can get your hands on some microfluidic tech, you probably already have: your inkjet printhead incorporates microfluidics!

This is one part of my Microfluidics Beginner’s Guide. Check out the rest of it and keep learning!

Reference:

ResearchBlogging.org Whitesides, G. (2006). The origins and the future of microfluidics Nature, 442 (7101), 368-373 DOI: 10.1038/nature05058