subject: Using Laboratory Centrifuge For Blood Cell Separation [print this page] Centrifugation is a primary method for processing blood because of its high throughput and versatility Whole blood can be easily and reproducibly fractionated into different components through differential centrifugation and selective removal. The blood cell separation can subsequently be used for their respective clinical and scientific applications and investigations. Aqueous density media have been used with centrifugation to separate and isolate different blood cell types. The dense cushion and low centrifugal force make possible the separation of non-damaged red blood cells (erythrocytes) and different types of white blood cells (granulocytes, lymphocytes and monocytes).
Laboratory centrifuge used for special needs of blood cell fractionation have rotors and labware that accommodate tubes for blood cell separation by gradient media. Good quality centrifuges provide enhanced versatility and performance with additional rotor and labware combinations.
Several commercially available gradient media kits with separation tubes of 12 to 15 mL size are commonly provided with the commercial kits. Larger 50 mL tubes can also be used for larger quantities of blood. Freshly drawn, anticoagulant-treated, whole blood should be processed within 2 hours. Defibrinated blood is obtained by shaking whole blood for 15 minutes with 2 to 3 glass beads to form a clot, and then the clot around the glass beads is removed by pouring the defibrinated blood into a fresh tube. Blood is commonly diluted 1:1 with PBS (phosphate buffer saline).
A typical process involves the following steps where separation is achieved by centrifugation. A temperature of 18C""26C should be maintained throughout the process (including the blood sample, the density medium-containing tubes, and centrifuges capable of maintaining that temperature range).Place gradient medium into the bottom of the microfuge tube. Then layer (diluted) blood carefully over the gradient reagent (consult the suppliers" guidelines for gradient and blood volume); avoid mixing.
In some kits, the tube is designed with two chambers (for blood and gradient medium) separated by a frit. The gradient medium is spun down (1,000 x g for 30 sec) to bring it below the frit. The blood sample is placed in the upper chamber.
This sample is centrifuged(blood layered on gradient). At the end of the spin, a Coasting (no brake) deceleration is recommended for better results as disturbance of sample separation is minimized. Low brake (slow deceleration) can also be chosen if reduced deceleration time is desired. After centrifugation, lymphocytes and monocytes can be found at the gradient""plasma interface, and erythrocytes along with granulocytes pellet to the bottom of the tube.
In a different gradient separation, leucocytes will separate into two bands; the upper contains mononuclear cells and the lower contains polymorphonuclear cells (PMNs). Longer centrifugation time will result in the PMNs migrating further downward. Erythrocytes are pelleted at the bottom.
