Phlebotomy Textbook - Chapter 12

Centrifugation

Centrifugation refers to specimen handling/processing during centrifugation. It causes the specimen to rotate at high speeds, thereby creating a centrifugal force that causes heavier elements to sink to the bottom of a test tube and lighter substances to stay on top of the specimen tube. During the spin, particles move with varying speeds down the tube depending on their size. Thus, solid elements (cells) flow to the bottom, and the liquid portion of the specimen (serum or plasma) stays at the top. The centrifugation time and speed are critical parameters that each manufacturer and laboratory establishes. Each laboratory should have procedures in place for the equipment that it uses for centrifugation (BOX 12-2). Although the procedures take a significant part of the turnaround time of the laboratory processing phase, it is important to comply with them because reduced centrifugation time can result in an incomplete barrier gel formation and residual blood cell elements in the plasma or serum, thereby causing analytical interference. Manufacturers' specifications generally indicate the speeds and times of centrifugation. FIGURE 12-8 describes centrifuges com-monly used in laboratories. The following are general guidelines for proper centrifugation: ■ As tubes are being loaded into the centrifuge, inspection of each one assures the tube has been filled to the correct fill line. If not, the specimen tube may be rejected. ■ Serum specimens should be allowed to clot before centrifugation. Complete clotting takes 30-60 minutes; however, be mind-ful that patients on anticoagulant therapy (heparin, Coumadin) and chilled specimens will take longer to clot. Incomplete clotting before centrifugation can leave fibrin residue in the serum, which would interfere with analyses.

Postcentrifugation

Postcentrifugation refers to specimen handling/processing after centrifugation and prior to removal of serum or plasma. Serum or plasma should be physically separated from cells as soon as possible and no longer than 2 hours from the time of collection (unless there is conclusive evidence that longer times do not affect specific test results.) The serum or plasma that is separated from the cells must be handled according to specified testing procedures, but in general, sera (plural form of serum) can remain at room temperature (22°C [71.6°F]) for testing, be refrigerated (2-8°C [35.6-46.4°F]), be stored in a dark place, or be frozen (at or below -20°C [-4°F]), depending on the prescribed laboratory method. All specimen refrigerators and freezers should be marked appropriately with biohazard labels. Many studies have been done to test stability of specimens at varying temperatures for varying time durations. Evidence suggests that separated serum/plasma should remain at 22°C for no longer than 8 hours. If testing cannot be completed within 8 hours, the specimen should be refrigerated. ■ Unless manufacturers' directions instruct otherwise, if testing is not completed within 48 hours, the separated serum/plasma should be frozen at or below -20°C. Frost-free freezers are not suitable for storage, because they automatically go through freeze/thaw cycles that can potentially activate coagulation factors. ■ Aliquot preparation occurs when the specimen must be subdivided into multiple tubes for testing on different instruments. The same identification information or code should be placed on each aliquot specimen

Precentrifugation

Precentrifugation refers to specimen handling/processing after collection and prior to centrifugation. For specimens that do require centrifugation, the general rule of thumb is that serum or plasma should be removed from cells as soon as possible and not exceed 2 hours from the time of collection. This means that processing should proceed without delay. ■ Anticoagulated specimens (plasma specimens) can be centrifuged immediately after collection. ■ Specimens without anticoagulant additives (serum specimens) should be clotted prior to centrifugation, which usually takes 30-60 minutes at room temperature (22-25°C [71.6-77°F]). If the specimen is not allowed to fully clot, fibrin strands can clog up analytical instruments and may cause erroneous results. ■ Clotting time is affected (often delayed) by anticoagulant therapy that the patient may be taking (Coumadin, warfarin, heparin, etc.). If clotting is incomplete, fibrin strands may have formed but the full clot might not form, so instrument malfunctions may occur. ■ Chilling the specimen will delay clotting. ■ Clotting may be accelerated by activators such as glass or silica particles, thrombin, and snake venom/thrombin. They may reduce clotting times to 15-30 minutes. ■ Because some analytes are photosensitive, they should be kept wrapped with aluminum foil or placed in an amber specimen container to shield the specimen from light for as much of the processing as possible. ■ Some laboratories still use the practice of removing the tube closure to "rim the tube" with a wooden applicator stick. This releases a blood clot attached to the tube closure or to the sides of the tube. However, this practice is not recommended because of the aerosol dangers, and because it may cause hemolysis. It is not necessary since there have been many manufacturer improvements in the tube/closure systems in recent years.

turnaround time (TAT)

The time it takes for a blood specimen to be ordered, collected, transported, processed, analyzed, and a result reported

Specimens that require transport at 37°C (98.6°F)

These specimens require either a commercially available controlled heat source or a heating block for transportation and handling purposes. Laboratory Tests: ■ Cold agglutinins ■ Cryofibrinogen ■ Cryoglobulins

A specimen should be protected from light for which of the following determinations? a. Bilirubin concentration b. Hemoglobin level c. Glucose level d. Blood cultures

a.

Which of the following analytes should be protected from light? a. Porphyrins b. Blood gases c. Cholesterol d. Glucose

a.

After blood collection, when should plasma be centrifuged? a. As soon as possible b. 10 minutes c. 30 minutes d. 60 minutes

a?

A critical laboratory value

also called critical value or alert threshold, is a test result that falls either higher or lower than critical limits established by each laboratory for specific tests. These results are taken seriously and may be life-threatening for the patient unless action is taken by the patient's physician.

Which analyte is photosensitive? a. Glucose b. Bilirubin c. Cholesterol d. Albumin

b.

Which of the following practices will likely cause hemolysis? a. Taking the top off the test tube b. Shaking the tube to mix the contents c. Gently mixing the tube d. Covering the tube with aluminum foil

b.

Which one of the following would be considered a preexamination/preanalytical error? a. Reporting a critical lab value on the wrong patient b. Testing the wrong aliquot for a glucose test c. Underfilling the aliquot container d. Changing a result without documenting it

c.

Which blood specimen should be chilled during a 45-minute transport? a. Porphyrins b. Cold agglutinins c. Cholesterol d. Blood gases

d

Which blood specimen should be kept warm during transport to the laboratory? a. Cholesterol b. Blood gases c. Porphyrins d. Cold agglutinins

d.

Serum should be removed from blood cells after which time period? a. 10 minutes b. 30 minutes c. 60 minutes d. 120 minutes

d.?

Laboratory Tests that need to be chilled

■ Ammonia ■ Blood gases ■ Catecholamines ■ Gastrin ■ Lactic acid ■ Parathyroid hormone ■ Pyruvate

Laboratory tests that are photosensitive

■ Bilirubin ■ Beta-carotene ■ Folate ■ Porphyrins ■ Vitamin A ■ Vitamin B6

The preexamination variables that are inherent in phlebotomy duties include:

■ Patient variables—Identification, age, gender, pregnancy, medications, fasting ver-sus nonfasting, diurnal variations, refusal to cooperate, patient unavailable, stress or anxiety, etc. ■ Transportation and tube handling variables—Correct filling and mixing of the specimen in the right tubes for individual tests, specimen leakage, tube breakage, excessive shaking, etc. ■ Specimen processing and storage variables—Adequacy of centrifugation, sample registration and distribution, delays in processing, contamination of the specimen, exposure to heat or light, etc. ■ Specimen variables—Hemolysis, inadequate volume in the tube, inadequate mixing of anticoagulant, etc.

Specimens that are photosensitive and should be protected from light

■ Photosensitive means that the substance is sensitive to light and will decompose after exposure to bright light. ■ Chemical constituents in blood that are light sensitive, such as bilirubin, will decompose if exposed to light. ■ Blood collected for light-sensitive chemical analysis should be protected from bright light with a commercially available amber/brown biohazard bag, an aluminum foil wrapping around the tube, or a transport container that does not allow light to enter (see Figures 12-4 and 12-5). ■ Recent studies have suggested that exposure to light may not affect folate and B vitamins, so follow your facility's proce-dures for protecting specimens as needed.

Plasma-based coagulation assays

■ Temperature fluctuations for coagulation tests should be avoided during transport. Use of ice or cold packs is not recom-mended because of possible cold activation of clotting factors and platelet disruption. ■ Ideally, transportation should occur within 1 hour of collection. However, acceptable time delays vary depending on the test ordered; for example, PT assays may be acceptable even after a delay of up to 24 hours. However, if monitoring unfractionated heparin therapy, the delay should not exceed 1 hour if collected in sodium citrate. Therefore, follow your facility's procedures for transport and handling coagulation specimens. ■ Specimens should be stored with a cap or stopper. ■ PT specimens can remain uncentrifuged or centrifuged with the plasma on top of the cells at room temperature (up to 24 hours). Specimens for APTT from patients who are not on heparin therapy can remain uncentrifuged or centrifuged with plasma on the cells at room temperature for up to 4 hours from the time of collection. If specimens contain unfractionated heparin, they should be kept at room temperature and centrifuged within 1 hour of collection. Again, follow your facility's procedures. ■ For molecular hemostasis assays, the ideal DNA extraction would occur from fresh specimens. However, anticoagulated, whole blood (EDTA or citrate) can be stored at room temperature for up to 8 days or at 2-8°C (35.6-46.4°F) before the DNA is extracted. Laboratories should also follow manufacturers' directions and provide up-to-date information to all personnel who are involved in transporting these specimens. ■ Coagulation specimens may be rejected if they are clotted, in the wrong additive, if the tube is overfilled or underfilled, if there are time delays, or if transported at the wrong temperature. Follow all procedures accordingly. ■ Acceptable time delays can vary depending on the specific coagulation test performed.

Specimens that are thermolabile and may require chilling

■ Thermolabile means that the substance is sensitive to higher temperatures, and specimens being tested for these sub-stances need to be chilled. ■ Chilling a specimen inhibits blood cell metabolism and stabilizes most constituents. ■ Chilling can be accomplished using commercially available products such as chilled packs or by the use of an ice water slurry (mixture of ice and water; do not use large chunks of ice because of the likelihood of freezing the specimen) (FIGURE 12-5). ■ Special note for blood gas specimens: Delivery speed is essential to prevent the loss of blood gases from the specimen. Each facility will have procedures for handling blood gas specimens that should be carefully followed and delivered quickly to the laboratory for analysis. ■ Some analytes are negatively affected by chilling, such as potassium (K). If a potassium test is ordered and collected in the same tube with other analytes that require chilling, it should be tested within 2 hours or collected in a separate tube. Chilling for more than 2 hours causes K to leak out of the cells, resulting in a false elevation. ■ Specimens for electrolytes should not be chilled.