How are the plasma and red blood cells separated using anticoagulated tubes?

Cell Salvaging Through Centrifugation and Washing Techniques

One of the simplest forms of autotransfusion is the use of a cell-salvaging system that uses aspiration and anticoagulation to collect shed blood and return it to the patient. The simplest products to perform this function include collection sets consisting of double-lumen tubes through which an anticoagulant (usually heparin or citrate-phosphate-dextrose) is mixed with shed operative blood, aspirated by negative pressure through a vacuum source, collected in a reservoir, and directly reinfused to the patient through a filter. Inherent problems with this technique include questionable quality of reinfused blood because of contamination with particulate matter aspirated from the field that includes bone fragments, fat particles, and suture materials. In addition, the anticoagulant remains present in the reinfusate. However, this technique is a relatively easy and quick means of returning lost blood in the event of unexpected acute blood loss.

Another form of autotransfusion uses specific machines that salvage and process shed blood and include a cell-washing step. The termcell saving has come to denote the process of autotransfusion that involves centrifugation of collected operative blood and processing with a wash solution, 0.9% NaCl, and reinfusing the product back to the patient. The basic operating principles found in autotransfusion include aspiration, anticoagulation, centrifugation, washing, and reinfusion. The ensuing discussion focuses specifically on the processes of cell washing and separation by centrifugation as autotransfusion methods.

The major components of any automated or manual device used for cell processing in autotransfusion are listed inBox 32.8. The process begins with the aspiration of blood from the surgical site together with an anticoagulant via a double-lumen line. The blood, together with other operative contaminants, including bone chips and adipose tissue, is then collected in a cardiotomy reservoir, functioning as the first filtration, with depth and screen filters ranging in size from 40 to 120 µm. A peristaltic pump then transfers the contents from the cardiotomy reservoir into a centrifuge bowl that has been specifically designed to separate blood according to specific particulate density. Centrifugation necessary for this separation process generally is between 4800 and 5600 rpm (Fig. 32.32). The volume of the bowl is an important characteristic of these devices because this capacity has a role in the minimum amount of shed blood required to obtain an acceptable hematocrit in the returned product. Some systems come with 125-mL bowls for use with small patients or when smaller amounts of shed blood are anticipated. The heavier RBCs are packed farthest from the axis of rotation, whereas the lighter plasma and crystalloid fractions remain closest to the center of the bowl. A wash mode is initiated when the centrifuge bowl has reached its optimal packed RBC level, with sterile physiologic saline pumped through the RBC layer, removing plasma-free hemoglobin, clotting factors, anticoagulant, and nonautogenous particles.

1.18.7.3 Centrifugation

Centrifugation has been used to separate colloids from aqueous solution on the basis of particle size and density. The samples are prefiltered to remove particulate material (by definition through a 0.45-μm filter paper) and then placed in centrifuge tubes. Samples usually undergo centrifugation at 25,000 rpm for a minimum of 45 min. This is repeated until the conductance and surface tension correspond to that of pure water.42 Again it takes protracted time scales to process samples, making the method unsuitable for marine and estuarine solutions where large sample volumes are required. However, it is suggested that centrifugation is far more effective than diafiltration and ion exchange at removing LMW molecules, and that centrifugation tubes are readily decontaminated and sterilized. The main disadvantage of centrifugation is therefore the limited sample capacity of the centrifuge.

12.1.6 Differential Centrifugation Sedimentation

DCS is used to measure the NP size distribution, where the NP size ranges from 2 nm to 80 μm. Major advantages of DCS are as follows: (i) ultrahigh resolution capability, (ii) measure peaks of different sizes, and (iii) also measure of small additional peaks, which can be compared with SEM. The study of Zeljkakrptic et al. on gold NP functionalized with organic ligands (peptides and PEG thiol) shows that DCS can be used to measure even the small thickness variation of as small as 0.1 nm on particle. The study of another group, Angela Jedlovszky et al., reveals that DCS is not capable to provide the quantitative structural data of protein-SPION complexes, but it can provide the valuable qualitative information (Jedlovszky-Hajdu et al., 2012). The study of Waczyk et al. interprets that DCS can measure the size distribution in semiqualitative way in a protein complex mixture (Walczyk et al., 2010; Kharazian et al., 2016).

Centrifugation (Bactofugation)

Centrifugation, sometimes called ‘bactofugation’ because the commercial equipment manufactured by Tetra Pak is marketed under the trade name Bactofuge™, uses centrifugal force of ∼9000 g to separate bacteria (and somatic cells) from milk. Separation is based on differences in the specific gravity (SG) of milk and bacterial cells. Milk has an SG of 1.028–1.038 g ml-1; bacterial spores 1.30–1.32 g ml-1; and vegetative bacterial cells 1.07–1.12 g ml-1. Thus, centrifugation is more efficient in removing spores than in removing vegetative cells from milk.

Typically, when operating in the optimum temperature range of 55–60 °C, centrifugation reduces the total bacterial count of milk by 80–90% (∼1-log cycle). However, it can remove 98–99.5% of anaerobic spore-forming microorganisms such as Clostridium and ∼95% of aerobic sporeformers such as Bacillus. A slightly higher reduction can be achieved by a second centrifugation. The gain in shelf life of refrigerated milk by centrifugation is about 4–5 days.

A major application of centrifugation, as for microfiltration discussed above, is in cheese manufacture where it is used to remove bacterial spores such as C. tyrobutyricum and C. butyricum, which can cause the ‘late blowing’ defect in cheese. Centrifugation can also be combined with lysozyme addition to reduce the level of nitrate added to inhibit Clostridium growth and prevent late blowing of cheese.

The use of centrifugation is also beneficial for removing spores in the production of UHT milk, whey protein concentrates, infant formulae, and milk powders. In these cases, application of sufficient heat to destroy spore-forming bacteria is not possible because of its effect on heat-labile milk components, especially whey proteins.

A more detailed coverage appears in the article PLANT AND EQUIPMENT | Centrifuges and Separators: Applications in the Dairy Industry.

6.7.3.2 Centrifugation

Centrifugation can be further classified into conventional and fluidized-bed modes. In conventional centrifugation, the cells are compacted against a solid surface as a result of the centrifugal force. This may be detrimental to cell health and also decreases the washing efficiency. The most common method of conventional centrifugation is a traditional bench-top centrifuge; this is not appropriate for clinical or commercial scale manufacturing because it is an open, nonscalable process. However, some other technologies incorporate closed, single-use disposables along with automation to conduct multiple rounds of centrifugation and bring residual levels below the required specification. Examples of such equipment are the CellSaver (Haemonetics, Braintree, MA), the COBE blood processor (TerumoBCT, Lakewood, CO), the Cytomate system (Baxter, Deerfield, IL), the CARR Centritech Lab III and CARR Centritech Unifuge (Pneumatic Scale Angelus, Clearwater, FL). The UniFuge represents a large-scale conventional centrifugation system in which the cell suspension is continuously fed into the single-use cylindrical chamber, where the cells are compacted against the wall while the clear supernatant is continuously removed. Once the cylinder fills with cells, the solids are discharged and the cycle can be repeated until all cells have been processed.

In counterflow centrifugation, the cells are maintained in a fluidized bed as a result of the flow of cell suspension being parallel but opposite to the direction of centrifugal force. The cells remain in the developing bed while residuals and clear supernatant are continuously removed. Two examples of counterflow centrifugation equipment are the Elutra from Terumo BCT (mainly used at smaller scale for separation of peripheral blood into its constituents) and the kSep400 and kSep6000 systems from KBI Biopharma. The kSep systems are based on automated, closed, and single-use technology with chamber capacity ranging from 100 mL to 6 L. The main advantage of counterflow centrifugation is that cells are not compacted against a solid surface and that multiple washes can be achieved in a relatively short period of time.

How can plasma and red blood be separated?

What procedure separates plasma from blood cells?

Which tube is used for plasma separation?

When blood is collected with an anticoagulant The whole blood separates into?