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Blood Separation and Centrifugation

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There is a relationship between the physical properties of blood; liquid viscosity, particle density, particle size, fraction of dissolved solids and the physical principles of centrifugation (gravitational force applied) that impact separation. Guidelines are provided to improve the efficiency of blood component separation using differential centrifugation.

Introduction

There is only so much plasma that can be obtained from a unit of whole blood. Total plasma volume is determined by the volume of blood collected and the donor percent hematocrit (% Hct). Figure 1 illustrates the theoretical range of plasma that can be recovered from a unit of whole blood.

Figure 1

Theoretical Total Plasma Volume Dependent on Donor % Hct and Collection Volume
Plasma Optimization: Theoretical Total Plasma Volume Dependent on Donor % Hct and Collection Volume 

Principles of Blood Separation

Blood separation is accomplished by sedimentation and can be defined as the partial separation or concentration of suspended solid particles from a liquid by gravity. The rate of sedimentation is a function of liquid viscosity, particle density, particle size, concentration of the solution (fraction of dissolved solids), and the force of gravity. Sedimentation rates can be calculated for any particulate fluid using Stokes Law of Sedimentation.iThis equation states that at any given “g-force”, the rate of sedimentation of a particle is directly proportional to its size and density and relative to the density of the suspension fluid. To accelerate sedimentation, the effect of gravity is amplified using “centrifugal force” provided by a centrifuge and can be many thousand times the force of gravity. 

Separation of cellular constituents within blood can be achieved by a process known as differential centrifugation. In differential centrifugation, acceleration force is adjusted to sediment certain cellular constituents and leave others in suspension. During the process of differential centrifugation of blood, the sample is separated into two phases: a pellet consisting of cellular sediment and a supernatant that may be either cellular or cell-free.

iStokes Law of Sedimentation
Vg = d2 (Þp - Þ1) / 18μ x G
where: Vg = sedimentation velocity, d = particle diameter, Þp = particle density, Þ1= liquid density, G = gravitational acceleration, μ= viscosity of liquid

Standardizing Centrifugation Terminology

Centrifugation terminology can be confusing. Abbreviations such as RPM, RCF, TCF and ACE™ are used to describe centrifugation conditions. As the discussion progresses on plasma yield optimization, it is important to clearly understand common centrifugation terminology. Standardization of centrifugation terminology will simplify the approach used later in this document to optimize centrifugation and improve plasma recovery. Centrifugation terminology is defined below.
  • Revolutions per Minute (RPM) – is the rotating speed of the rotor arm within the centrifuge during centrifugation. Example: 5000 RPM. The centrifuge is spinning at 5000 Revolutions per Minute.
  • g – Force or “g” – is a unit of acceleration equal to the force of gravity.
  • Relative Centrifugal Force (RCF) – is the force during centrifugation that moves a particulate away from the center of rotation. It is expressed as multiples of the earth's gravitational field (g). Since RCF includes rpm in the calculation, it changes as the rpm changes during the centrifugation cycle. During acceleration the RCF increases, when the rpm set point is attained, the RCF is constant and during deceleration the RCF decreases. Therefore, the RCF calculated during the centrifugation cycle reflects the force applied at any particular instant in time. The formula to calculate relative centrifugal force in “g” is as follows:

          RCF (g) = (rpm)2 x (1.118 x 10-5) x radius of rotor (cm)
          RCF (g) = (rpm/1000)2 x 28.38 x radius of rotor (inches)
  • Total Centrifugal Force (TCF) – is a calculation for determining the total applied centrifugal force over the complete centrifugation cycle and has units of g•s. Whereas RCF is the force applied at a given time in the centrifugation cycle, TCF is the Total Force Applied over the complete time and uses RCF (g) x time (s). TCF (g•s) =RCF (g) x time (s)
  • Accumulated Centrifugal Effect1 (ACE) – is a calculation for determining the total applied centrifugal force over time and uses speed (RPM) and time (s). This calculation can be programmed on most Sorvall™ centrifuges as the “integrator function” or ∫ω2dt.
While centrifugation protocols are frequently defined as RPM and time, this practice can introduce significant variability from one center to another or one centrifuge to another when trying to reproduce the same process. Expressing the force of centrifugation using RPM alone does not take into consideration radius of the rotor or centrifuge load. The variability of centrifugation can be reduced by standardizing the terms of acceleration to specify the acceleration (RCF: g) or total centrifugal force (TCF: g x s) or accumulated centrifugal effect (ACE) that is to be applied to the sample, rather than specifying revolutions per minute and time. Calculation of RCF is dependent upon the radius of the centrifuge rotor used. The same centrifuge manufacturer with different rotors can produce different acceleration forces (defined as multiples of “g” or the earths’ gravitational force). An example of the difference in RCF for the same RPM settings is shown in Table 1.

Table 1

Relative Centrifugation Force Values for Various Centrifuge Rotors
Centrifuge Manufacturer Beckman Jouan Sorvall Sorvall Sorvall Sorvall Sorvall
Rotor Model JS4.2 KR4 22 H6000A HBB-6 H12000 HLR-6 H4000
Rotor Radius (cm) 25.4 28.0 26.1 25.5 29.7 25.8 23.1
RPM Relative Centrifugal Force
4200 5009 5522 5139 5029 5861 5080 6483
5000 7099 7826 7284 7127 8307 7200 13022
Difference in g-force applied 2090

2304

2144 2098 2446 2120 6539

Table 1 shows the impact of using RCF and RPM nomenclature interchangeably. In this example the assumption that centrifugation of blood components at 5000 RPM is equivalent to an RCF of 5000 x g could subject blood components to elevated g-force and therefore compromise the component quality and the efficiency of component separation.

RPM does not equal RCF and must not be used interchangeably

Defining the time of centrifugation is also important in standardizing centrifugation protocols. Time may be defined in minutes of acceleration at a given RCF or it may be incorporated in the final calculation of either total centrifugal force2 or accumulated centrifugal effect. Both TCF and ACE calculations are used to standardize the total effect of centrifugation and use a calculation of either speed and time (ACE) or RCF and time (TCF) to calculate the area under the curve (AUC). An example using area under the curve to calculate TCF for centrifugation is shown in Figure 2.

Figure 2

Calculating Total Centrifugal Force Using Area under the Curve

Plasma Optimization: Calculating Total Centrifugal Force Using Area under the Curve

Fraction 1 2 3 4 5 6 7 8 9 Total
Time(s) 0-45 78 111 152 416 442 462 480 576 576
Fractional AUC (RCF x time) 6548 24024 62486 156251 1320000 99606 38270 13104 13968 1.73 x 106 g.s

As shown in Figure 2, using AUC to calculate TCF is as simple as multiplying the RCF x time (s) for each incremental fraction (depicted in the graph as fractions 1-9) and adding the individual fractions that make up the curve.

A similar method of using time and RPM to calculate area under the curve and associated ACE settings using the integrator function ∫ω2dt is well described1.The benefits of using the ACE function are to standardize centrifugation by improving run-to-run reproducibility. The setting compensates for differences in acceleration associated with differences in rotor load, voltage fluctuations, loss of instrument calibration, and environmental factors like extreme temperatures2.

General Overview of Centrifugation for Blood Banking

There are three differential centrifugation processes used to separate whole blood into blood components for transfusion. These are: soft or light spin, hard or heavy spin, and buffy coat spin processing conditions. The soft spin centrifugation profile uses low centrifugal force (slower speed) to allow slower sedimentation of the lighter constituents in whole blood. The soft spin processing condition produces a plasma component that contains a large fraction of the platelets (platelet-rich plasma) and a softly packed red cell component. The buffy coat layer is located at the interface that separates the platelet-rich plasma from the packed red cells, and contains the majority of the white blood cells as well as some of the larger more dense platelets. The soft spin process is typically used to prepare platelets, packed red cells, and plasma from a whole blood component. 

Hard spin component processing uses high centrifugal force (faster speed and longer centrifugation time) and produces compacted red cell product and low cellular content, high volume plasma. In this case, the buffy coat layer contains most of the blood leukocytes and platelets. If at this stage the buffy coat layer is separated from the plasma and red cells and is subjected to another round of soft spin centrifugation, a buffy coat platelet product can be produced as is currently performed in Canada and many European countries. In the United States, the AABB recommends centrifugation conditions for the production of blood components as outlined in Table 2.

Table 2

AABB Recommended Centrifugation Conditions3for Component Separation
  Component Prepared Spin Conditions
Heavy Spin Red Cells 5000 x g, 5 minutes
Cell-free Plasma/Cryoprecipitate 5000 x g, 7 minutes
Light Spin Platelet-rich Plasma 2000 x g, 3 minutes

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