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Centrifugation in the Clinical Laboratory
Mar-Apr 2013 - Vol. 2 No. 2 - Page #6

The use of centrifuge technology is critical in the clinical laboratory setting for the separation of liquid and solid components. In laboratories performing biochemical analyses on body fluids, centrifuges are routinely used to separate blood cells from serum/plasma, to separate sediment from urine, to measure the volume fraction of erythrocytes in blood (the hematocrit), and to separate bound from free components in protein binding and immunoprocedures. Less routinely, centrifugation is used for separation of lipoproteins in reference procedures for their measurement, separation of cellular components, and separation of DNA fragments.1

In today’s clinical chemistry laboratory, high capacity horizontal head or swinging bucket units, wherein the specimen tubes are spun at a 90° angle to the shaft of the rotor, are the most common centrifuges. These devices centrifuge the vast majority of tubes at a speed of 3500 rpm for 10 minutes. In addition, temperature-controlled centrifuges—horizontal head or swinging bucket centrifuges that maintain a temperature of 4°C—are used to preserve the integrity of thermolabile analytes, which can be adversely affected by the heat generated by most devices during the centrifugation process.

Centrifugation for the Blood Bank and Coagulation
The blood bank and coagulation departments also are dependent on consistent and accurate centrifuge operation in order to separate serum/plasma from cellular components. The blood bank spins gel cards for antibody screening, cell washings, and tube typing. Separation of cellular constituents within blood is achieved through the process of differential centrifugation—a process where centrifuge 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.2 High-capacity horizontal head or swinging bucket centrifuges are also used in the coagulation department to obtain platelet-poor plasma. There are specific centrifuge parameters that must be adhered to in order to obtain the correct analyte results in the processed samples and these parameters are specified by collection tube manufacturers. In certain situations, those parameters may be analyte specific. An example of an analyte-specific requirement is double spinning of tubes for Protein C and S in the coagulation department. In general, the recommended centrifuge speed for chemistry testing is 3500 rpm for ten minutes; for coagulation testing, the spin speed is 3500 rpm for seven minutes.

Centrifugation Variables
At The Valley Hospital in Ridgewood, New Jersey, the clinical laboratory uses four centrifuges in the chemistry department and two in the coagulation department. In both areas, these devices are used continuously to process a large number of tubes throughout the day. The chemistry and coagulation departments centrifuge about 2,000 to 2,500 tubes per day. The peak centrifuge time occurs between 5AM and 11AM. During this time, approximately 75% of the workload comes into the laboratory from the hospital and outreach facilities. Although these six devices are used constantly, they also serve as backup devices in the event of an operation failure in one centrifuge. Fortunately, this technology is fairly durable and should last at least seven to ten years, even when subjected to constant use.

Regardless of the scope of use, the most important variables that must be accounted for are centrifuge speed, spin time, and temperature within the device. For example, improper speed or force—measured in rpm or relative centrifugal force (RCF)—or shortened spin times, will cause faulty separation of the serum/plasma from the cellular components (eg, RBCs, WBCs). Proper preventive maintenance of centrifuges is critical, as faulty or failing devices can lead to an improper or incomplete layering of the gel matrix between the serum/plasma and the cellular components, which can cause the serum/plasma to remain in contact with those cellular components. This contact can cause pre-analytical errors such as decreased glucose levels or increased potassium levels. Also, high temperatures will destroy the integrity of thermolabile analytes.

Operational Safety and Training
Most clinical centrifuges have several built-in safeguards against device failure. In our experience, failures generally occur only at the start of a run due to tube placement imbalances (see Figure 1). If such a load-balancing failure occurs, the device will stop automatically. When failure occurs, an alarm sounds or flashes to alert the operator of the problem.



To maintain proper functioning, our biomedical engineer checks and cleans all centrifuges twice a year. If a failure occurs during the interim, the biomedical engineer is contacted and the device is taken out of service until cleared for use. Centrifuges should be replaced when they require frequent and extensive maintenance to keep them operational.

Proper staff education on the efficient and effective use of centrifuges can be accomplished in several ways. One option is hands-on training during the clinical rotation in medical technologists’ laboratory curriculum. Another option is on-the-job training of new operators. When placing tabletop or standalone centrifuges in the laboratory, the devices should be easily accessible to staff members for loading and unloading of the tubes. A major advantage of tabletop models is the ability to have storage space below the table.

Conclusion
The safety and efficiency of modern clinical centrifuges continues to improve with many manufacturers and suppliers seeking to reduce the time it takes for the device to reach the designated speed as well as reduce the time required to stop the centrifugation process. Most centrifuges that are interfaced with laboratory hardware/software are part of analytical systems that have front-end specimen processing tracks. In such systems, specimen collection tubes are placed on a track that transports the tubes to a centrifuge. After centrifugation, the tubes are returned to the track for transport to the analyzer for testing. The speed and dependency that centrifuges bring to the lab make them an invaluable technology as we work to automate more practices in the lab.

Reference

  1. Lauritzen M. Quantities and units for centrifugation in the clinical laboratory. J Automat Chem. 1992;14(3):93-96.
  2. Blood Separation and Centrifugation. Plasma Optimization Guide: Improving Plasma Yields from Whole Blood Donations. Pall Medical. Available at: http://www.pall.com/pdfs/Medical/10.3422_Plasma_OG%281%29.pdf
  3. Frothingham R. Centrifugation without a balance tube. Am Biotechnol Lab. 1999;17(3):84.

Jean I. Jarzabek, MS, MT(ASCP), CC(NRCC), is a senior chemistry technologist at The Valley Hospital in Ridgewood, NJ. She has 40 years of clinical chemistry experience and shares her expertise as a faculty member at the hospital’s School for Clinical Laboratory Scientists. Jean received her BA in zoology and MS in biochemistry. She is active in the American Association for Clinical Chemistry, having served as chairperson of the New Jersey section and as member of several national level committees.


Table 1.
Algorithms for Balanced Centrifuge Rotor Loading

Requirements

  • The rotor has n positions, such that n is divisible by 6, or
  • If less than 6 an even number of positions
  • The number of samples is between 2 and n, but not equal to n-1
  • Tubes and sample volumes are identical

Algorithm A. Sample number is even:

  • Load pairs of tubes in opposite positions

Algorithm B. Sample number is odd:

  • Load three tubes at equal intervals in the rotor, forming an equilateral triangle
  • Load the remaining tube in pairs in opposite positions

Examples of thermolabile analytes include:

  • Ammonia
  • Lactic acid
  • Renin
  • Coagulation factors

 

 

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