Separating Blood into Cellular and Non-Cellular Components
Nov 30, 2021
A key diagnostic step in disease detection is analyzing blood components. Accurate separation of blood cells from plasma, which holds proteins and other constituents, is crucial in many assays.
First introduced in the late 1980s, lateral flow devices, also referred to as lateral flow immunoassays, are a simple, rapid, and cost-effective point-of-care diagnostic tool. Today, the technology and applications have evolved and there is increasing demand for the use of whole blood as the sample matrix.
A lateral flow immunoassay is based on the chromatography-like migration of a labeled analyte through multiple materials, including a sample pad, conjugation pad, detection membrane, and absorbance pad, ending in the visible result of an immobilized captured reagent.
Being able to separate a sufficient quantity of plasma from cells is a central tenet to maximizing performance of these devices. Generally in a laboratory setting, plasma can be separated from a whole blood sample by mechanical methods using sedimentation or centrifugation, but this requires additional instruments for sample processing that are not available in an at-home setting.
In lateral flow devices, the liquid sample to be analyzed is placed on a sample pad. This part of the test system performs several important functions—even distribution of the sample and direction of its movement to the conjugation pad at a certain rate as well as acting as a filter to remove unwanted elements of the liquid biosubstrates, such as blood cells.
Challenges of blood separation
From a lateral flow, rapid test point of view, blood separation means that the cellular components are to be retained at the point of sample application while the plasma with all its components, such as antibodies, and other proteins that could include proteins shed from viruses or bacteria, hormones, salts, etc., is expected to migrate through the different parts of the test system, according to Klaus Hochleitner, PhD, global lead technical product specialist diagnostics, Cytiva.
The biggest challenge in this approach is the limited loading capacity of the available sample pads. “This has two consequences. First, there is a risk of red blood cell breakthrough upon overloading and/or lysis of red blood cells due to mechanical damage, poor sample handling, or insufficient pad pre-treatments, which typically leads to an unspecific red background on the detector membrane that results in a poor signal-to-noise ratio,” explains Hochleitner. “Secondly, the volume of moving plasma is typically too small to make the test systems function properly. The addition of another buffer that pushes the plasma through the system is required, which, unfortunately, increases the complexity of the test, and also the possibility of user mistakes.”
Novel solutions are being developed to address these challenges. One approach is the modification of the standard blood-separation matrices with proteins that bind specifically to blood cells, such as lectins or specific antibodies. “This increases the loading capacity, and it provides a specific retention that reduces the risk of blood cells flowing further onward into the test system,” says Hochleitner. “But the downside is the limited shelf life of such matrices as compared to the simple blood separation pads currently available as well as the additional costs of these materials.”
The second approach is in the lateral flow cassette design. “Several conventional blood separators can be stacked on top of each other in a sort of funnel that is mounted on the application port of the lateral flow cassette to allow for the addition of larger blood volumes,” continues Hochleitner. “Although the relative percentage of recovered plasma decreases as the number of stacked pads increases, this approach can work nicely.”
The need for advanced blood separation systems for lateral flow devices will accelerate as more point-of-care tests using whole blood are developed, especially those for diagnosing infectious diseases. According to Hochleitner, another requirement will be safe methods to obtain blood samples from the patient with a volume of a couple of 100 μL. A new generation of blood separation materials, along with test design modifications, is expected to make rapid tests even more user-centric and convenient.
However, most if not all experienced users know how to work with existing easy-to-obtain sample pads and accept the associated drawbacks.
Hochleitner believes that working with pad manufacturers in a joint development effort on customized solutions so that the blood separators are designed for a specific test system makes more sense. This opens the door to the development of more convenient tests for users that also produce more reproducible and reliable results due to reduced variability of the blood separation process.
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