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Blood Separation Techniques: Performing Blood Cell Isolation from Whole Blood Samples

March 2020

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Blood Separation Techniques: Performing Blood Cell Isolation from Whole Blood Samples

Each blood cell type has its own unique purpose and function and separating the various blood components allows scientists to analyze specific cell types. For example, human leukocytes can be used to study immunological functions such as cytokine production and surface marker expression. Other blood components can be used to detect diseases.

Once separated, blood cells can be further isolated into cell subsets.

Components of Whole Blood

Whole blood contains red blood cells, white blood cells, platelets, and plasma. The optimal blood separation technique will depend on which component is being isolated.

Plasma

The largest portion of blood is plasma, a protein-rich substance made of 90% water, lipids, salts, and hormones. Plasma makes up around 55% of total blood volume in the human body and performs several necessary functions. Plasma transports blood cells, coagulates blood, regulates the body’s water, and carries out a multitude of other tasks. Lacking essential plasma proteins such as albumin or immunoglobulins can cause serious health issues.

The other three blood components are suspended in plasma.

Red Blood Cells (RBCs)

Red blood cells (RBCs), also called erythrocytes, transport oxygen, and carbon dioxide throughout the body providing tissues with oxygen for cells to use and sending carbon dioxide to the lungs for expulsion. These cells are the second most abundant portion of whole blood, making up roughly 44% of the overall composition. While RBCs are some of the most vital cells in the human body, they are also one of the most common contaminants of cell samples.

White Blood Cells (WBCs)

White blood cells (WBCs), also known as leukocytes, sustain the body’s immune system by attacking infectious cells and foreign invaders. Less than 1% of a total blood sample are WBCs. They are slightly larger than RBCs and carry out specialized roles. WBCs are further categorized into mononuclear cells (lymphocytes and monocytes) and polymorphonuclear cells (neutrophils, eosinophils, and basophils).

Platelets

Platelets, or thrombocytes, act as “first responders” at the site of injury to form clumps that help stop bleeding. These cells also typically make up less than 1% of blood composition and are smaller than both red and white blood cells. When the body receives a laceration or puncture of some kind, platelets can combine with a protein substance called fibrin to prevent further damage by making a blood clot.

Blood Separation Techniques

To properly study blood cells, the individual cell type must be isolated to remove contamination interference. Some blood separation techniques rely on the different densities of whole blood components for separation while others, such as Akadeum’s BACS, rely on antibody-based sorting.

Plasma Separation from Blood Centrifuge

Blood is usually separated from plasma through centrifugation. The physical force from continuous revolutions pushes the denser, heavier particles to the outer edges of the sample resulting in three layers of different densities: RBCs, a mixture of WBCs and platelets, and plasma. The addition of a density separation medium can also cause the isolation of mononuclear cells and granulocytes from human blood.

Centrifugation and other blood separation techniques may leave behind residual RBCs, which can extend sort times and impede subsequent cell separation.

Blood Apheresis Machine

Apheresis is a process in which blood is removed from a patient or donor, sorted into specific cell types for some sort of treatment or manipulation, then reintroduced back into the patient. This can help treat individuals with blood disorders or certain autoimmune diseases.

Some researchers use a blood apheresis machine to separate whole blood into its components. A blood apheresis machine receives blood that is extracted from a patient’s body and sorts it into plasma, platelets, WBCs, and RBCs. This device can be helpful when consistently dealing with large samples of whole blood, but for smaller experiments or T cell isolation assays, a blood apheresis machine is not cost-effective.

RBC Separation from Whole Blood Sample

RBCs can be separated or isolated from whole blood in a variety of ways, but some are more efficient than others. While it might take extra money and time to preserve the extracted RBCs, certain methods (such as RBC depletion) simply remove the contaminants without damaging the surrounding blood cells. At Akadeum, our response to RBC contamination is RBC depletion with our innovative microbubble technology.

RBC Depletion

Depletion occurs when a single cell type is removed from a biological sample (i.e. red blood cells). Human peripheral blood mononuclear cells (PBMCs) are often used to study immunological functions such as cytokine functions and expression on surface markers. High quantities of RBCs can create background interference in a PBMC sample and reduce the accuracy of downstream assays. Their removal is key to achieve full functionality. The removal of RBCs from PBMCs qualifies as RBC depletion.

Akadeum offers an RBC Depletion Kit that further prepares a cell sample to be separated into specific cell subsets.

White Blood Cell Isolation from Whole Blood

WBCs are responsible for a variety of functions in the human body. Scientists can study the behaviors of WBCs to get a better understanding of how the body naturally responds to foreign pathogens and harmful substances.

White Blood Cell Isolation Protocol

Depending on the chosen blood separation technique, the strategies for preparing a whole blood sample and extracting target cells will differ. For many WBCs, whole blood must be sorted into a PBMC sample then further isolated into individual blood cell subtypes.

Preparing Peripheral Mononuclear Cells (PBMCs)

PBMCs are white blood cells with round nuclei, which include T cells, B cells, NK cells, dendritic cells, and monocytes. To separate these from plasma and RBCs, many scientists use centrifugation with a density gradient medium. After centrifugation, the PBMCs will be grouped together above the density gradient in a liquid layer for easy collection. PBMC preparation allows scientists to study only cells of interest or further isolate specific cells from a smaller cell population with no RBC contamination.

Purpose of Blood Separation

Each blood cell type has its own unique purpose and function. Separating the various blood components allows scientists to analyze specific cell types. Some blood components can be used to detect diseases, while others can provide a more comprehensive understanding of the human immune response. Blood separation allows researchers to study only the target cell population without any interference from other cell types.

Strategies for Reducing Unwanted Contamination

Contamination from RBCs and other residual blood cells can be prevented or solved through RBC depletion OR by following protocols and using high purity cell isolation methods. There is no need for RBC depletion if the blood separation technique is accurate enough. Therefore, it could be very beneficial to use an antibody-based sorting technique that targets specific cell subsets from the beginning.

Buoyancy Activated Cell Sorting for Sample Preparation

Buoyancy activated cell sorting, or BACS, uses streptavidin-coated tiny glass bubbles to target surface receptors on unwanted cells. Once the bubbles attach to those cells, they float them to the top for removal. After all the labeled cells have been removed, the enriched sample remains untouched at the bottom of the mixture for downstream assays. BACS is beneficial for experiments that require additional tests or experiments because it maintains cell health and physiology. The gentle and buoyant nature of the bubbles can effectively isolate delicate cells with high purity and accuracy.

How Akadeum Life Sciences Uses BACS to Supplement Blood Separation

BACS can be used for blood sample separation in more ways than one. The T cell and B cell isolation kits can be used to purify specific cell types out of blood samples quickly and efficiently.

If you’re using a different cell separation method, Akadeum’s Human Red Blood Cell Depletion Microbubbles can be used to reduce contamination beforehand–eliminating as much as 97% of RBCs within 10 minutes without damaging cell sample viability.

When working with lymphocyte isolation or RBC removal from whole blood samples, Akadeum offers a cost-effective, time-saving solution with our patented microbubble technology. Not convinced? Download our app note on the Removal of Red Blood Cells From Human Peripheral Blood Mononuclear Cell Samples Using Akadeum Human Red Blood Cell Depletion Microbubbles for more information on blood separation techniques, or talk to a scientist today.

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