Specimen Collection, Preparation and Handling

Preparing the Vacuum Tube

This article is presented as a guide for trained venipuncture technicians, or phlebotomists, and is not intended to train individuals in venipuncture technique.

Assembling Supplies.

Assemble the following supplies: lab coat, gloves, labels, needle holder, tourniquet, appropriate tubes, gauze or cotton balls, alcohol sponge, and adhesive strip. (See Figure 1). The aseptic method of collecting and transporting a blood specimen works on the principle of a vacuum tube for drawing blood. A standard double-pointed needle or multisampling needle (both disposable) may be used for venipuncture. Ordinarily, a 21- or 22- gauge needle is used. A small bore, sharp needle causes minimum patient discomfort; 22- or 23- gauge is the smallest bore (or lumen) size recommended to avoid hemolysis. A needle length of 1 to 1? inches permits an angle of entry that will not pierce both vein walls and enter tissue.

FIG 1	FIG 2

When more than one blood specimen is required, multisampling needles and vacuum tubes make blood collection simpler and more efficient. (See Figure 2). A tiny rubber sleeve automatically closes when the vacuum tube is removed from the holder, preventing leakage and loss of blood when the tubes are being changed.

• 1. Open the single or multisampling needle package; do not remove the needle sheath (sterile shield). (See Figure 3).
• 2. Thread the needle into the holder and tighten it firmly. (See Figure 4).

FIG 3	FIG 4

• 3. Slide the collection tube into the holder, carefully pushing the tubes forward until the needle touches the stopper.
• 4. Carefully push the tube forward until the top edge of the stopper meets the guideline on the holder. Let go. The tube will retract below the guideline. Leave it in that position. This step embeds the full point of the needle in the stopper without puncturing it, preventing blood leakage on venipuncture and the premature loss of vacuum. (See Figure 5).

FIG 5

During venipuncture, do not have the patient clench and unclench the first repeatedly. This will cause hemoconcentration (a local increase in red blood cells). Also, never leave a tourniquet on the arm for more than 2 minutes without releasing it. This can cause discomfort to the patient and may also cause hemoconcentration.

Preparing the Puncture Site.

After securing the tourniquet and reaffirming your selection of the best vein, both by sight and palpation, proceed as follows.

• 1. Except when blood alcohol is ordered, swab the site with a sterile alcohol sponge (70% alcohol) in a circular motion, inside to outside, to push contaminants away from the puncture site. If you are going to palpate the vein again after prepping the puncture site, remember that your finger should also be prepped with a separate alcohol sponge or by immersion in alcohol. (See Figure 6).

FIG 6

• 2. Allow the puncture site to air dry after swabbing, or dry the site with gauze or cotton. If alcohol is not allowed to dry, it may contaminate the specimen. If the arm is dry, you will avoid stinging the patient at venipuncture.

Note: When drawing blood, please follow all approved venipuncture procedures recommended for use by recognized organizations and/or in accordance with state regulations involving phlebotomy practices.

Considerations for Single and Multisampling Collection.

If only a single collection tube is required, remove the entire assembly from the arm when the vacuum is exhausted. Place a piece of dry sterile gauze over the needle and withdraw the needle carefully.

FIG 7	FIG 8

• 1. When multiple specimens are required, remove the first collection tube from the holder as soon as blood flow ceases, invert the first tube to prevent clotting, and gently insert the second tube into the holder. Puncture the diaphragm of the stopper by pushing the tube forward and initiating vacuum suction. (See Figure 7). Remove and invert each successive tube after it is filled. When all samples have been drawn, remove the entire assembly from the arm. (See Figure 8). Do not recap, cut or bend any needles; dispose of them in a safe, responsible manner.

Note: When multiple specimens are drawn from a single venipuncture, the following order is recommended: 1) sterile blood culture tubes, 2) not additive clotting tubes (red), 3) coagulation tubes and tubes containing citrate (blue), 4) serum-separator tubes, 5) tubes containing heparin (green or royal blue), 6) tubes containing K3 EDTA (lavender), 7) tubes containing acid citrate dextrose (ACD yellow), and 8) tubes containing sodium fluoride and potassium oxalate (gray).

• 2. After drawing blood, observe proper venipuncture techniques to prevent continued bleeding and/or hematoma.

Note: If the blood has to be mixed with an anticoagulant, this must be done immediately after drawing. You can do this quickly while the patient’s arm is elevated. Mix blood with anticoagulant thoroughly, using a rolling wrist motion and by inverting the tube gently five or six times. (See Figure 9).

FIG 9	FIG 10

• 3. Label tubes immediately after confirming all necessary information with the patient. (See Figure 10).

Syringe Transfer Technique in Venipuncture

With the syringe technique, venipuncture is accomplished without direct connection to the collection tube. To ensure specimen integrity, follow these steps.

• 1. Use disposable plastic syringes and disposable needles. For most laboratory specimens, using 20 mL plastic syringes will allow the withdrawal of adequate specimen. Generally, the needle should not be smaller than 21-gauge.
• 2. If glass syringes are used, it is essential that the barrel and plunger be absolutely dry. Small amounts of moisture can cause hemolysis. If the glass syringe has been autoclaved, it should be oven dried before use. Air drying techniques are usually not satisfactory.
• 3. After the blood is collected by syringe, push the needle immediately through the vacuum tube stopper, and allow the blood to be drawn into the tube. Place the vacuum tube in a rack before pushing the needle into the stopper.

Note: When multiple specimens are prepared from syringe-filled tubes, the following order is recommended: 1) sterile blood culture tubes, 2) coagulation tubes and tubes containing sodium citrate (blue), 3) tubes containing K3 EDTA (lavender), 4) tubes containing heparin (green or royal blue), 5) tubes containing acid citrate dextrose (ACD yellow), 6) tubes containing sodium fluoride and potassium oxalate (gray), and 7) nonadditive clotting tubes (red or serum-separator).

Blood Preparation Procedures

There are two important guidelines to follow when submitting blood specimens. For some tests, such as chemistry procedures, fasting samples are often the specimen of choice. Also, because hemolysis and lipemia interfere with many procedures, please submit samples that are as free from hemolysis and lipemia as possible.

Preparing Serum

Serum Preparation From Red-Top Tube.

Follow the steps below when preparing a serum specimen for submission.

• 1. Draw whole blood in an amount 2 ? times the required volume of serum so that a sufficient amount of serum can be obtained. The 10-mL red-top tube will yield approximately 4 mL serum after clotting and centrifuging. The 15-mL red-top tube yields approximately 7 mL serum. Label the specimen appropriately.
• 2. Place the collection tube in the upright position in the rack, and allow the blood to clot at room temperature for no longer than 30-45 minutes. (See Figure 11). If clotting fails to occur within 30 minutes, notify the physician.

FIG 11	FIG 12

• 3. After allowing to clot to form 20-30 minutes, insert the tube in the centrifuge, stopper end up. Operate the centrifuge for 15 minutes at the speed recommended by the manufacturer. Do not allow prolonged centrifugation as this may cause hemolysis. When using a bench-top centrifuge, employ a balance tube of the same type containing an equivalent volume of water. The tube stopper should remain. (See Figure 12).
• 4. Turn the centrifuge off and allow it to come to a complete stop. Do not stop it by hand or brake. Remove the tube carefully without disturbing the contents.
• 5. Remove the stopper and carefully aspirate all serum from cells, using a separate disposable pipette for each tube. Place the tip of the pipette against the side of the tube, approximately ? inch above the cell layer. (See Figure 13). Do not disturb the cell layer or carry any cells over into the pipette. If cells do enter the pipette, recentrifuge the entire specimen

FIG 13	FIG 14

• 6. Transfer serum from the pipette into the transport tube. (See Figure 14). Inspect the serum for signs of hemolysis and turbidity by holding it up to the light. Be sure to provide the laboratory with the amount of serum specified.
  
• 7. Label the tube carefully and clearly with all pertinent information or bar code. Unless otherwise indicated, serum samples may be sent at room temperature. When multiple tests requiring frozen serum are ordered, a plastic transport tube should be prepared for each test.
  
• 8. When frozen serum is required, place the plastic transport tube(s) immediately in the freezer compartment of the refrigerator. Notify your professional service that you have a frozen sample to be picked up. A separate frozen sample must be submitted for each test requiring a frozen sample.

Serum-Separator Tubes.

Serum-separator (mottled red/gray or cherry red-top) tubes contain clot activator and gel for separating serum from cells but include no anticoagulant. Adhere to the following steps when using a serum-separator tube. Do not use serum-separator tubes to submit specimens for which tricyclic antidepressant levels, Direct Coombs’, Blood Group, and Blood Types are requested. There are other times when serum-separator tubes should not be used. Always consult the test description prior to collection.

• 1. Draw whole blood in an amount 2 ? times the required volume of serum so that a sufficient amount of serum can be obtained. The 10-mL red-top tube will yield approximately 4 mL serum after clotting and centrifuging. The 15-mL red-top tube yields approximately 7 mL serum. Label the specimen appropriately.
• 2. Gently invert the serum-separator tube five times to mix the clot activator and blood.
• 3. Place the collection tube in the upright position in the rack, and allow blood to clot at room temperature for no longer than 30-45 minutes. (Clots usually form in 20-30 minutes.)
• 4. After allowing the clot to form 20-30 minutes, insert the tube in the centrifuge, stopper end up. Operate the centrifuge for 15 minutes at the speed recommended by the manufacturer. Do not allow prolonged centrifugation as this may cause hemolysis. When using a bench-top centrifuge, employ a balance tube of the same type containing an equivalent volume of water.
• 5. Turn the centrifuge off and allow it to come to a complete stop. Do not stop it by hand or brake. Remove the tube carefully with out disturbing the contents. Inspect the barrier gel to insure that it has sealed the serum from the packed cells. Also, examine the serum for signs of hemolysis and turbidity by holding it up to the light. Be sure to provide the laboratory with the amount of serum specified.
• 6. Make sure the tube is clearly labeled with all pertinent information or bar code.
• 7. If a frozen specimen is not required, it is not necessary to transfer serum to a plastic serum tube.
• 8. When frozen serum is required, always transfer the serum (using a disposable pipette) into a separate, clearly labeled plastic transport tube. Place the tube immediately in the freezer compartment of the refrigerator, and notify the professional service representative that you have a frozen specimen to be picked up. Never freeze a glass serum-separator tube. Submit a separate clearly labeled plastic transport tube for every test requiring a frozen sample. Unless otherwise indicated, serum samples may be sent at room temperature.

Plasma Preparation.

When plasma is required, follow these steps.

• 1. Always use the proper vacuum tube for tests requesting special anticoagulant (eg. EDTA, heparin, sodium citrate, etc) or preservative.
• 2. Tap the tube gently to release additive adhering to the tube or stopper diaphragm. (See Figure 15).

FIG 15

• 3. Permit the vacuum tube to fill completely. Failure to fill the tube will cause an improper blood-to-anticoagulant ratio and yield questionable test results.
• 4. To avoid clotting, mix the blood with the anticoagulant or preservative immediately after drawing each sample. To ensure adequate mixing, slowly invert the tube five or six times using a gentle wrist rotation motion.
• 5. Immediately centrifuge the specimen for 5 minutes. Do not remove the stopper.
• 6. Turn the centrifuge off and allow it to come to a complete stop. Do not stop it by hand or brake. Remove the tube carefully with out disturbing the contents.
• 7. Remove the stopper and carefully aspirate plasma, using a separate disposable Pasteur pipette for each tube. Place the tip of the pipette against the side of the tube, approximately ? inch above the cell layer. Do not disturb the cell layer or carry any cells over into the pipette. Do not poor off; use transfer pipette.
• 8. Transfer the plasma from the pipette into the transport tube. Be sure to provide the laboratory with the amount of plasma specified.
• 9. Label all tubes clearly and carefully with all pertinent information or bar code. All tubes should be labeled with the patient’s full name or identification number as it appears on the test request form or affix bar code. Also, print on the label the type of plasma submitted (e.g., “Plasma, Sodium Citrate,” Plasma, EDTA,” etc).
• 10. When frozen plasma is required, place plastic transport tube(s) immediately in the freezer compartment of the refrigerator, and notify your professional service representative that you have a frozen specimen to be picked up. Never freeze glass tubes.