Author: Klaus Hochleitner.
Many healthcare professionals use whole blood tests to check for the presence of biomarkers quickly and simply. Common examples include molecular testing for infectious diseases in places without a laboratory and portable test kits for cardiac markers used at home or in an ambulance.
Both red and white blood cells can interfere with lateral flow tests when you are looking at extracellular markers. Red blood cells are strongly colored and active in redox reactions, influencing the result and readout. White blood cells contain DNA, which can also affect the sensitivity of rapid test assays.
Efficiently separating plasma from blood cells can improve the sensitivity and reliability of your diagnostic tests, which is key in developing a reliable assay. However, there are several common issues which might prevent effective blood separation. Read more on these challenges and the potential remedies for each below.
Blood separation challenge 1: What is hemolysis?
Hemolysis occurs when red blood cells burst open and release their contents — most importantly hemoglobin — into the blood plasma. When this happens, the plasma turns red, making it more difficult to judge the readout reliably.
One common reason for hemolysis is the use of old samples or stress on the cells because of handling. The risk of hemolysis reduces when fresh blood is applied to the pad, also known as a blood separation membrane, with minimal handling steps.
Using an unsuitable type of glass fiber can also cause hemolysis. Small diameter glass fibers can apply high stress to a cell membrane, effectively cutting the red blood cell open. If you think fiber diameter might be causing hemolysis, try using a filter with a larger diameter fiber to reduce stress and risk of damaging cell membranes.
The type of wash buffer used in the filtration process also influences the risk of hemolysis. Wash buffers contain surfactants, which have the potential to break cell membranes, and salts, which cause cells to swell or shrink if concentrations are not optimal. Isotonic buffers with mild surfactants will minimize the risk of the wash buffer damaging the cells.
Blood separation challenge 2: Red blood cell breakthrough
A similar but separate effect to hemolysis is red blood cell breakthrough; both cause the plasma to retain its red color after separation. Common causes for cell breakthrough include leakage around the edges of a separator and the use of blood volumes that exceed a separator’s capacity.
If your initial tests show cell breakthrough, in some cases an easy solution can be to either reduce the sample volume or increase the volume of the blood separation pad. Having sufficient compression along the edges of the pad and making sure that cells cannot leak around the edges will also minimize breakthrough.
Blood separation challenge 3: Analyte binding
The glass fibers of a filter can sometimes bind analytes on their surface. The hydrophobic nature of glass causes some proteins and nucleic acids to stick to the fibers through hydrophobic interaction.
One way to avoid sticky fibers is by coating them with a polymer that makes the matrix more hydrophilic. Polyvinyl alcohol (PVA) is a common hydrophilic coating, and PVA-coated filter materials, such as Fusion 5, provide highly hydrophilic surfaces that do not interact with most analytes.
Blood separation challenge 4: Uneven blood separation
Inhomogeneities within the filter matrix can cause uneven separation of a sample. Liquid flowing faster in some parts of the pad than in others can lead to different test run times and inconsistencies in results.
This uneven flow might result from the design and construction of the test or subtleties in the assay protocol. Making sure to select a highly homogeneous filter material and providing clear instruction on the method and position of sample application can help achieve consistent blood separation.
How to choose the correct type of filter
There are a range of filter materials suited to blood separation. The most appropriate type of filter for your application often depends on capacity — and therefore the thickness — required for the expected sample.
Examples of depth filter materials for different volumes include:
- LF1, for volumes between 10 and 15 µL
- MF1 and Fusion 5, for volumes between 15 and 50 µL
- VF2 and GF/DVA, for volumes larger than 50 µL
Unsure of which filtration material to use?
Our team of experts are trained in troubleshooting filtration and can advise on which materials are most suitable for your assay. They can help identify the cause of and solution to any issues you might be having in diagnostic assay development. Contact your local Cytiva representative to discuss your needs.