Except for the glass red-topped tube, which has no additives, all tubes contain one or more additives.
Additives include anticoagulants to prevent clot- ting, clot activators to promote it, thixotropic gel to separate components, and preservatives and inhibi- tors of various cellular reactions to maintain the integrity of the specimen.
Any tube containing an additive must be inverted and mixed well immediately after removal from the adapter. Gently turning the tube over, and then back upright again, equals one inversion. You should not shake the tube because this will cause hemolysis.
The exact number of inversions needed varies by tube type; most need five to eight inversions. Many tubes are coated inside with silicone to prevent blood from adhering to the wall of the tube and to slow the clotting process.
Anticoagulants
Anticoagulants prevent blood from clotting. So- dium or potassium ethylenediaminetetraacetic acid (EDTA) binds calcium, thereby inhibiting the co- agulation cascade. Other additives that bind calcium include sodium citrate, potassium oxalate, and sodium polyanetholesulfonate (SPS). Another anti- coagulant, heparin (linked with sodium, lithium, or ammonium), inhibits clotting by preventing the conversion of prothrombin to thrombin. Having the correct ratio of blood to anticoagulant is important, so you must take care to fill the tube completely.
Anticoagulants must be mixed well by gently and repeatedly inverting the tube.
The choice of anticoagulant is determined by the tests to be done. EDTA preserves blood cell in- tegrity well, prevents platelet clumping, and is compatible with blood staining, but it interferes with coagulation studies. Citrate is used for coagu- lation studies. SPS is used for blood cultures be- cause it inhibits certain immune system components that could otherwise destroy blood-borne bacteria and neutralizes antibiotics that the patient may be taking. Heparin is preferred for plasma chemistry determinations and for blood gas determinations.
Potassium oxalate, combined with sodium fluoride or iodoacetate, is used for glucose determination.
FLASHBACK
You learned about blood clotting in Chapter 7.
Sodium fluoride inhibits glycolysis and can be used for glucose determination. Glycolysis is a cellular
reaction used to harvest energy from glucose. Lithium iodoacetate is an alternative antiglycolytic agent.
Clot Activators
Clot activators promote coagulation. Clotting in plain serum tubes takes about 60 minutes, while clotting in serum tubes with clot activators takes only 30 minutes.
Thrombin directly increases clotting and is used for stat serum chemistry determinations or if the patient is on anticoagulants (e.g., often prescribed to prevent a recurrence of stroke). Inert substances such as glass or silica promote clotting by providing more surface area for platelet activation. Other inert substances are sili- ceous earth, clay, and Celite. Clot activators may be adhered to the side of the tube; therefore, the sample must be inverted five times to allow the blood to come in contact with the activator. Some inert substances may interfere with certain tests (e.g., blood bank procedures) and therefore cannot be used.
Polymer Gel
Polymer gel, also called thixotropic gel, is an inert, synthetic substance whose density is between that of cells and that of blood serum or plasma. When the specimen is centrifuged, the gel becomes a liquid and moves between the lower cell layer and the upper se- rum or plasma layer. It hardens again after standing and forms a barrier between the two layers, thus pre- venting contamination and allowing easy separation.
For instance, in the light green–topped tube used for determining potassium, the gel prevents the plasma from being contaminated by potassium released by red blood cells (RBCs). (Thixo- means “related to touch”
and refers to the gel’s ability to liquefy when shaken.) Color-Coded Tops*
Each combination of additives is distinguished by a different colored top. However, different manu- facturers may use slightly different color-coding schemes. In some situations, the requisition indi- cates the color of tube to use; in other cases, the name of the test is specified, and you need to deter- mine the correct tube. This information is found in the laboratory’s directory of services.
Yellow, Sterile
Tests: Blood culture
Additives: SPS to inhibit complement and phagocy- tosis
Specimen: Whole blood
Note: Use this tube to recover microorganisms caus- ing blood infection.
Light Blue
Tests: Coagulation tests Additives: Sodium citrate Specimen: Plasma
Notes: Fill the tube completely to maintain the ratio of nine parts blood to one part sodium citrate (e.g., 4.5 mL of blood to 0.5 mL of citrate). Invert three or four times.
Red, Plastic or Glass Tube
Tests: Chemistry, serology, blood bank
Additives: Clot activators in plastic, none in glass Specimen: Serum
Notes: Plastic tubes should be inverted gently five times. Blood collected in a red-topped plastic or glass tube takes 60 minutes to clot. Serum is separated by centrifugation after clotting.
Lavender
Tests: Complete blood count (CBC), sedimentation rate, routine immunohematology testing
Additives: EDTA Specimen: Whole blood
Notes: Dipotassium (K2) EDTA is spray-dried onto the sides of the tube. This is the form preferred CLINICAL TIP
Any tube with an additive should be inverted gently and repeatedly to mix the contents immediately after collection.
Do not shake tubes, as this can cause cells to become damaged, resulting in hemolysis.
*Colored tube tops are courtesy and © Becton, Dickinson and Company.
by the Clinical and Laboratory Standards Insti- tute (CLSI), as described later. Tripotassium (K3) EDTA is in liquid form, and disodium (Na2) EDTA is in powdered form.
Pearl
Tests: Viral loads
Additives: Polymer gel with EDTA Specimen: Plasma
Gold BD Hemogard Closure or Red-Gray
Tests: Most chemistry tests
Additives: Clot activators, polymer gel Specimen: Serum
Notes: These are also called serum separator tubes (SSTs), “stat” tubes, or tiger-speckled. Invert such tubes gently five times. Clotting requires a mini- mum 30 minutes.
Gray
Tests: Lactic acid measurement, glucose tolerance test, fasting blood sugar (FBS), blood alcohol levels
Additives: Antiglycolytic agent (iodoacetate or so- dium fluoride) that preserves glucose, perhaps the anticoagulant potassium oxalate or heparin Specimen: Plasma
Notes: Iodoacetate preserves glucose for 24 hours;
sodium fluoride preserves glucose for 3 days.
Black
Tests: Sedimentation rate (though often done with a lavender tube instead)
Additives: Buffered sodium citrate Specimen: Whole blood
Note: Fill the tube completely to maintain the ratio of four parts blood to one part citrate.
Green
Tests: Stat and routine chemistry tests, ammonia, elec- trolytes, arterial blood gases (ABGs), depending on heparin additive
Additives: Sodium heparin, lithium heparin, or am- monium heparin
Specimen: Plasma
Light Green or Green–Gray
Tests: Stat and routine chemistry tests Additives: Heparin, polymer gel Specimen: Plasma
Note: These are also called plasma separator tubes (PSTs).
Orange BD Hemogard Closure or Yellow–Gray
Tests: Stat chemistry Additives: Thrombin Specimen: Serum
Notes: This tube type allows for a 5-minute clotting time. It is used for patients on anticoagulant therapy.
Royal Blue
Tests: Toxicology, trace metals, nutritional analysis Additives: Heparin, EDTA, or none
Specimen: Plasma or serum
Notes: These tubes are chemically clean, and the stop- pers are specially formulated to prevent the release of small amounts of materials that could contami- nate the sample and give erroneous test results.
Tan
Tests: Lead analysis Additives: Heparin Specimen: Plasma
Note: The tube is formulated to contain less than 0.1 mcg/mL of lead.
Yellow, Nonsterile
Tests: Human leukocyte antigen (HLA) studies (paternity testing and tissue typing)
Additives: Acid citrate dextrose Specimen: Whole blood
Notes: The dextrose nourishes and preserves RBCs, and the citrate is an anticoagulant.
Pink
Tests: Blood bank compatibility test Additives: K2 EDTA
Specimen: Plasma or whole blood
Note: This tube is similar to the standard lavender- topped tube, but its closure and label meet the standards set by the American Association of Blood Banks.
Good technique can reduce this risk somewhat (dis- cussed in more detail in the next chapter); however, it cannot eliminate it entirely. For this reason, the CLSI has developed a set of standards dictating the proper order of draw for a multitube draw (Figure 8-11). The order is the same for syringe samples as for direct filling from a multisample needle. The order-of-draw standards have under- gone several revisions within the past decade, and not all institutions have adopted the most recent set of standards (termed H3-A6).
It is important for you to follow the order of draw used at your institution, even if it differs from the order given here.
1. Blood culture tubes (which are sterile) are drawn first. This prevents the transfer of unsterilized material from other tubes into the sterile tube.
AVOID THAT ERROR!
It’s the third day on the job for Maria Hernandez. She consults
her requisition for the next patient, and sees that he needs a coagulation test. Maria assembles her materials, including tourni- quet, swabs, bandage, and a lavender tube. She enters the pa- tient’s room, asks him to state his name, checks the wrist band, and proceeds with the draw. Afterward, she labels the tube, makes sure bleeding has stopped, and thanks the patient as she leaves. But as she closes the door, she stops, a look of panic on her face. What did she do wrong? And what should she do now?