EXERCISE 13:SPECIMEN PROCESSING

Skills:25 points

Objectives

  1. Explain the basic use and principle of how a centrifuge works.
  2. Name five (5) types of centrifuges and explain their specific uses.
  3. List nine(9) general rules for the operation of the clinical centrifuge.
  4. Identify two (2) important factors for proper balance of tubes in a centrifuge.
  5. Explain the dangers associated with operating an imbalanced centrifuge.
  6. Distinguish between whole blood, serum and plasmafor testing purposes.
  7. Explain how EDTA and heparin prevent blood from coagulating.
  8. Define and recognize hemolysis, icterus, and lipemia and explain their effects on laboratory tests
  9. State five(5) items that must be included on a tube labeled for an aliquot.
  10. State the different types of plasma used in testing.
  11. Define pre-analytical error and identify the phlebotomist role in preventing them.
  12. List 14 reasons that a specimen would be rejected by the laboratory.
  13. State the action which must be taken when a sample is rejected by the laboratory.
  14. Explain the importance of verifying the blood specimen identification with the requisition slip.
  15. Demonstrate proper accessioning of laboratory specimens by correctly filling in the accessioning log sheet.
  16. Demonstrate appropriate specimen processing by comparing requisitions with specimens and noting any discrepancies, problems with specimen suitability, and/or missing specimens.
  17. State the resolution of any problems noted on the Specimen Accession Log.
  18. Correctly label a transfer tube for serum or plasma aliquot.
  19. Safely and accurately separate serum or plasma from cells using appropriate PPE.

CENTRIFUGES

Centrifuges are instruments that use centrifugal force to separate suspensions in liquids. The most frequent laboratory use of the centrifuge is to separate the heavier cellular components of blood from the liquid serum/plasma so that it may be used for testing.

Centrifuges vary in size, capacity, configuration and speed capability. Centrifuges that have a fixed angle head will hold and keep the sample at an angle during the spinning process. Swinging head or swinging bucket centrifuges have a hinge that allows the sample to swing outward. How the blood sample appears depends on whether it was spun in a fixed head or swinging bucket type of centrifuge.

Common types of clinical laboratory centrifuges

  1. Clinical centrifuge is the name given to tabletop models which can be used for urinalysis or serum separation. These models come in either fixed head or swinging bucket configurations and usually have a speed capacity of up to 3000 rpms (revolutions per minute). Typical clinical centrifuges are capable of holding tubes with volumes ranging from 5 to 50 mL.
  2. A serofuge is a small tabletop centrifuge primarily used in blood bank or serology to spin serological tubes. These centrifuges can be fixed head or swinging bucket type.
  3. Microcentrifuges or microfuges are powerful compact table top centrifuges becoming increasingly popular. They are designed to spin special microtubes of 1.5 mL capacity at high speeds, usually about 12,000 rpm.
  4. The microhematocrit centrifuge is a variation of the microfuge, used to spin anticoagulated blood collected in capillary tubes in preparation for the measurement of hematocrits. Microcentrifuges are usually of the fixed head configuration.
  5. Other types of centrifuges include high-speed centrifuges which rotate at speeds up to 20,000 rpm and ultracentrifuges which rotate at speeds over 50,000 rpm. These centrifuges are often specially equipped with temperature control capabilities. Theses centrifuges are more commonly found in research laboratories. High-speed/ultracentrifuges are usually of the swinging bucket configuration.

Operation of Centrifuges

The following are general rules; always follow manufacturer’s instructions for your instrument’soperation and maintenance.

  1. Preventive Maintenance. Centrifuges must be evaluated and undergo preventative maintenanceregularly. The routine checks of revolutions per minute using either a tachometer or a stroboscope are absolutely essential for consistent results that meet quality control standards. The timer must also be checked against a calibrated timer for proper results. All routine and preventive maintenance activities, as well as repairs, must be recorded in the appropriate Quality Assurance manual.
  2. ALWAYS wear gloves when handling specimens. Follow the facility protocols for wearing additional appropriate PPE, such as disposable lab coats, protective eyewear, safety goggles, or face shields.
  3. NEVER spin specimens that do not have a stopper or cap. Spinning open-top specimens creates potentially harmful aerosols.
  4. ALWAYS use tubes appropriate for centrifugation in the type of instrument you are operating.
  5. NEVER operate a centrifuge on an uneven or slanted work surface. Centrifuges must be kept on a level and firm work bench.
  6. NEVER operate a centrifuge with the lid open. Do not open the centrifugelid while the rotor is moving. Although a ‘safety shutoff’ switch will be triggered if the centrifuge’s lid is opened, the rotor will continue to spin until friction eventually stops it.
  7. NEVER put your hands into the centrifuge until it has come to a complete stop, or try to stop it with your hand.
  8. ALWAYS balance the contents of the centrifuge before operating. Running a centrifuge with an unbalanced load could permanently damage the instrument AND poses a safety risk to you and those around you.
  9. Balance each tube in terms of size and volume. For every tube placed in a centrifuge, there must be a balance tube of identical type and must have an identical volume. For example, you would balance an SST tube with another SST tube. Most labs have a rack of balance tubes, which consist of the various tube used by that lab with water in them. These are used to balance the load.
  10. Balance the load across the rotor head.The rotor is the part of the centrifuge which holds the tubes and rotates during the operation of the centrifuge. Specimens that have been matched up in terms of size and volume are placed opposite from each other across the rotor.

TYPES OF BLOOD SAMPLES

Whole blood is composed of all cellular elements; red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs) suspended the liquid component, plasma. Adult blood has about 40% cellular elements and 60% liquid In tests requiring a ‘whole blood sample’, the blood is mixed with an anticoagulant at the time of collection, kept in a mixed or suspended state, and is NOT spun down. The most common laboratory test on a whole blood sample is the complete blood count (CBC). Other examples of whole blood tests include the Westergren sedimentation rate (ESR), Reticulocyte count, arterial and capillary blood gases.

Serum is the liquid expressed from clotted blood (blood drawn into a tube with no anticoagulant additive). Blood is allowed to clot and fibrinogen, along with some of the other coagulation factors, is used up in the formation of the clot. Serum, therefore, does not contain fibrinogen or most of the other coagulation factors. Serum is the preferred specimen for most chemistry, blood bank and serology tests because fibrinogen, which interferes with many tests, is removed.

Plasma is the liquid present in anticoagulated blood and contains all the coagulation factors, except one. Because plasma contains most of the clotting factors, it tends to be somewhat hazier than serum. Plasma is used for stat chemistries and coagulation studies.

Most anticoagulants, including EDTA, act by binding or chelating calcium, a necessary component of the coagulation process. The lack of available calcium prevents the blood from clotting.

Heparin is a naturally occurring anticoagulant which acts as an anti-thrombin.Thrombin is another essential component of the coagulation mechanism. The effects of heparin are relatively short-lived compared to other anticoagulants. After about 48 hours, blood drawn in heparin will begin to clot.

SPECIMEN PROCESSING

The following are general guide lines for processing patient specimens.

Chemistry Specimens

Serumspecimens are collected in Red, Gold, Red/Black, SST, or Royal Blue with a red label. Prior to centrifugation, these tubes must be allowed to clot completed, which usually takes 30 – 60 minutes.

Plasma specimens are collected in Green, Green/Black, PST, Tan, Brown, or Royal Blue with a green or lavender label. These tubes may be centrifuged as soon as they arrive in the processing area.

Immunohematology (Blood Bank)

Serumspecimens will be in Plain Red tubes. Plasma specimens will be Pink or Lavender tubes.

Regardless of specimen type, the tube is delivered to Immunohematology and processed by that department.

Hematology Specimens

Plasma specimens for coagulation testing are collected in Light Blue. These may be centrifuged as soon as they arrive in the processing area.

Whole Bloodspecimens forhematology are collected in Lavender or Black. These specimens are never centrifuged, but are placed on a rocker in the Hematology department to keep the specimen properly suspended.

Microbiology

Blood Culturesand any other specimen for microbiologyare delivered to the department for processing.

SERUM AND PLASMA APPERANCE

Once the patient sample has been centrifuged, observe its appearance. Generally it should be clear to hazy and some shade of pale yellow to yellow in color. Any usual color or appearance should be noted and may cause the specimen to be rejected for testing. The following are important appearance variations you must be aware of.

1. Hemolysis – A sample is said to be ‘hemolyzed’ when the serum / plasma has a red or reddish color. Hemolysis results when red cells rupture releasing the hemoglobin molecules. Gross hemolysis, where the serum or plasma appears bright red, affects most lab tests and the specimen should be recollected. Some tests will be affected by even slight hemolysis, when the serum or plasma is even slightly pink. Examples of tests that are affected by slight hemolysis include the chemistry potassium test and enzymes LDH (lactate dehydrogenase) & AST (aspartate aminotransferase).

If you find samples that are hemolyzed, check the laboratory / departmental procedure manual to see what effect it will have. The sample may need to be re-collected.

Hemolysis usually occurs when the venipuncture is traumatic, i.e., vein collapses, needle is moved several times, or negative pressure causes hemolysis of the fragile red cells. In most cases it is possible to avoid hemolysis by obtaining the blood sample with a ‘clean, non-traumatic stick’.

2. Icterus – The sample is said to be ‘icteric’ when the serum or plasma is deeply yellow, amber or even brownish due to liver disease, damage or excessive red cell breakdown inside the body. Patients with icteric samples may have hepatitis and are said to be jaundiced. Like hemolysis, icterus can affect many lab tests, but, unfortunately, recollection of the specimen would not improve the serum or plasma appearance. However, appearance should be noted on the lab report - "Serum icteric". In addition, icteric plasma or serum samples must be handled with extra caution because of the risks posed by the hepatitis.

3. Lipemia – A ‘lipemic’ sample has a milky appearance to the serum or plasma. Slight milkiness may be caused when the specimen is drawn from a non-fasting patient who has eaten a heavy meal. A heavy milky appearance occurs in rare cases of hereditary lipemia. As with icterus, the appearance should be noted on the lab report - "Serum lipemic". If lipemia is present and determined to be due to a non-fasting specimen, the specimen must be recollected. Lipemic specimens are avoided by drawing specimens from fasting patients.

For color pictures of hemolyzed, icteric and lipemic samples, please refer to the Equipment Review for Lab Practical at

ALIQUOTING THE SPECIMEN

After the sample has been spun down, the liquid serum / plasma must be transferred into another tube. The transfer tube in which the aliquot of serum or plasma is placed must be labeled BEFORE the aliquot is added to the tube. The label MUST include the following:

  1. Two patient identifiers:
  2. Patient name
  3. Other unique identifier (Examples: date of birth, lab accession number, medical record number, etc.)
  4. Date and time of collection.
  5. Initials of the individual aliquoting the specimen.
  6. Specimen type: EDTA plasma, citrate plasma, heparinized plasma, sodium fluoride plasma, serum, etc.
  7. Special information such as trough or peak drug level.

Take great care not to put the wrong specimen in the wrong tube. One way to avoid this devastating error is to work with only one patient specimen at a time.

Serum or Plasma Tubes WITHOUT GEL: The tube is centrifuged and the serum or plasma is carefully pipetted off into another clean, properly labeled test tube with a clean, disposable pipette. Care must be taken not to remix plasma specimens or contaminate either the serum or plasma with red blood cells.Red cells present in a serum or plasma sample will alter the results of many laboratory tests.

Serum or Plasma Tubes WITH Gel: During centrifugationthe gelmoves above the clot to form a physical barrier between the serum or plasma and the clot or red cell layer. The serum or plasma can then be poured off without contamination by red blood cells.

SPECIMEN STORAGE

If the specimen is to be stored in the refrigerator or freezer, the tube should be tightly capped or tightly sealed with Parafilm©. If the specimen is to be frozen, the ideal freezer is a true laboratory freezer which maintains constant temperature. Regular kitchen refrigerator freezers are generally frost free and go through freeze and thaw cycles, which may adversely affect the specimen.

ACCESSIONING SPECIMENS

Every lab has a procedure to accession specimens in the lab. Patient names and identification numbers are logged in on a computer or on a log sheet or book along with the test(s) to be performed. This accessioning serves as a record of tests done and is a convenient way to check if the specimen has actually arrived in the lab for testing. It is extremely important that all patient information is correctly transcribed, that includes spelling of the patient's first and last names and the correct identification numbers. Remember, most lab errors are clerical in nature. Examples of these types of errors include: misspelling of the patient name, and transposing identification numbers.

PRE-ANALYTICAL ERROR AND SPECIMEN REJECTION

Because the goal of the laboratory is to turn out accurate results, any problem with a specimen that compromises the quality or integrity of the results often means that the sample must often be recollected. The following is a list of some of the reasons samples will be rejected / or will need to be recollected:

  1. Labeling errors:

a. Name misspelled or wrong name.

b. Identification number does not match the requisition’s number.

c. Date of collection, time of collection and/or phlebotomist’s initials are missing.

  1. Unlabeled tubes.
  2. Wrong tube was collected.
  3. Patient was not fasting (when test required a fasting specimen).
  4. Timed specimen not collected at correct time.
  5. “Quantity not sufficient” (QNS) –specimen collected had an inadequate volume.
  6. Specimen hemolyzed.
  7. Anticoagulated specimen has clots in it.
  8. Improper transport (time, temperature, light exposure).
  9. Outdated (expired) supplies were used.
  10. Contaminated specimens.

Stand your ground and reject improperly labeled and/or collected specimens.

Resources and References

  1. Lab Manager: “6 Safety Tips for Operating a Centrifuge”.
  2. wikiHow – to do anything. “How to Use a Centrifuge”. (Be sure to watch the video.)
  3. “Using a Centrifuge”, Dr. Peter Darben, SPARQ-ed Coordinator, University of Queensland Diamantina Institute.

EXERCISE 13: SPECIMEN PROCESSING

Procedure

Your instructor will give you a sample tube(s) and requisition, and will explain the procedure using this sample.

Once the instructor has finished explaining the sample, you will be given a bag of specimens with requisitions. You should find requisition slips and tube samples for 3 different patients.

Although not every tube in the bag will have a true blood sample within, for the purposes of this lab you will process them as though they do, and as though all are properly filled.

  1. Using the Specimen Accession Log from the last page of this lab, fill in the date and time columns with today’s date and the current time.

2. Compare the names and identification numbers on EACH tube with the names and identification

numbers on the requisition slip. After each tube is evaluated place upright in a test tube rack.

  1. Write the patient's name and identification number in the appropriate column on your form.
  2. If any errors are identified, note the problem on your "Specimen Accession Log"
  3. Describe the action which must be taken to resolve the problem.
  4. List each test ordered on the requisition and the tubes received in the appropriate columns. If a problem is identified list the problem and the resolution.
  5. Make sure that you have the correct specimen for each test ordered.
  6. Note on the log if specimens are missing or inappropriate specimens were received.
  7. Describe the action which must be taken to resolve the problem, i.e. patient must be redrawn.
  8. Blue –top tubes (sodium citrate) are collected for coagulation studies (PT, PTT, etc.) It is very important that these tubes are filled to the appropriate amount (as indicated on the side of the tube). Once in the lab, the plasma must be efficiently harvested and separated from the cells. Often these tubes are centrifuged in the serofuge. There will be one specimen in your bag that has a sample that will require aliquoting.
  9. If a centrifuge is available in your lab you may be required to centrifuge your specimen.
  10. Make sure each tube has one directly opposite from it with identical size and blood volume. If no match is found create a balance tube.
  11. Once the centrifuge is full; spin for 7 - 10 minutes.
  12. Label a test tube for each specimen to be separated with the following information
  13. Patient's name
  14. Identification number or DOB
  15. Specimen type (refer to procedure)
  16. Date and time of collection
  17. Your initials
  18. Special labeling, i.e., peak, trough, etc.
  19. Carefully separate the serum or plasma from the original tube into the labeled tube with a clean pipette making sure not to contaminate the serum or plasma with red blood cells. If the red blood cells are resuspended the tube must be respun.
  20. Parafilm©or cap the top of each tube.Instructor will demonstrate this technique.
  21. Once you have finished, have your instructor check your work for errors before returning any tubes or requisitions to the bag.

EXERCISE 13:SPECIMEN PROCESSINGSTUDY QUESTIONS