Some of the common considerations affecting all types of specimens include:

• Failure to label a specimen correctly and to provide all pertinent information required on the test request form
• Insufficient quantity of specimen to run test or QNS (quantity not sufficient).
• Failure to use the correct container/tube for appropriate specimen preservation.
• Inaccurate and incomplete patient instructions prior to collection.
• Failure to tighten specimen container lids, resulting in leakage and/or contamination of specimens.
• Failure to maintain the specimen at the appropriate temperature requirement.

The most common serum preparation considerations include:

• Failure to separate serum from red cells within 15-30 minutes of venipuncture.
• Failure to allow clot specimens to clot before centrifugation.
• Hemolysis: red blood cells broken down and components spilled into serum. Causes and prevention are discussed under the section on hemolysis.
• Lipemia: cloudy or milky serum sometimes due to the patient's diet (discussed under the section on lipemia).

The most common considerations in the preparation of plasma include:

• Failure to collect specimen in correct additive.
• Failure to mix specimen with additive immediately after collection.
• Hemolysis or red blood cell breakdown.
• Incomplete filling of the tube, thereby creating a dilution factor excessive for total specimen volume (QNS).
• Failure to separate plasma from cells within 15-30 minutes of venipuncture for those specimens requiring this step.
• Failure to label transport tubes as “plasma.”
• Failure to indicate type of anticoagulant (eg, “EDTA,” “citrate,” etc).

The most common urine collection considerations include:

• Failure to obtain a clean-catch, midstream specimen.
• Failure to refrigerate unpreserved specimen or store in a cool place.
• Failure to provide a complete 24-hour collection/aliquot or other timed specimen.
• Failure to refrigerate unpreserved specimen or store in a cool place during collection period (ie, 24 hours).
• Failure to add the proper preservative to the urine collection container prior to collection of the specimen.
• Failure to provide proper mixing of specimen with urine preservative.
• Failure to provide sufficient quantity of sample to meet minimum fill line on preservative transport container.
• Failure to provide an appropriate collection container and to refrigerate specimen when bacteriological examination of the specimen is required.
• Failure to tighten specimen container lids, resulting in leakage of specimen.
• Failure to provide patients with adequate instructions for 24-hour urine collection.
• Failure to divide specimen into separate containers for tests with such requirements.
• Failure to provide a 24-hour urine volume when an aliquot from the 24-hour collection is submitted.

In general, grossly or even moderately hemolyzed blood specimens may not be acceptable for testing. Hemolysis occurs when the red cells rupture and hemoglobin and other intracellular components spill into the serum. Hemolyzed serum or plasma is pink or red, rather than the normal clear straw or pale yellow color.

Most cases of hemolysis can be avoided by observing the steps listed.

1. For routine collections, use a 21- to 22-gauge needle. (On occasion, however, it may be necessary to use a 23-gauge needle for patients from elderly and pediatric populations with small or difficult veins.)
2. If there is air leakage around the needle or loss of vacuum in the tube, replace the vacuum tube.
3. If you are using your own collection equipment instead of the vacuum tube technique, use only clean, dry, sterile needles, syringes, and tubes.
4. Collect blood in room temperature containers unless the specimen requirement specifies otherwise.
5. When there is difficulty accessing a vein or when a vacuum tube fills too slowly due to a difficult venipuncture, damage to the red blood cells may result. Correct by collecting a fresh tube when blood flow is established or select another puncture site and, using sterile/unused equipment, collect a second specimen. Also, a blood pressure cuff will reduce trauma to fragile red blood cells.
6. Do not remove the needle from the vein with the vacuum tube engaged. This applies to both the last tube collected during a routine venipuncture and to tubes collected during a difficult procedure.
7. Premature removal of the tube causes a rush of air to enter the tube, which may result in damage to the red cells.
8. Be as gentle as possible, drawing the blood evenly. Too much pressure in drawing blood into a syringe or forcefully ejecting blood into a collection tube from a syringe may damage red cells.
9. Allow collection site to dry after cleaning. Alcohol used to clean the puncture site may cause contamination in a tube.
10. Do not collect a specimen in a hematoma.
11. Allow specimen to clot completely before centrifuging.
12. Do not centrifuge the specimen for a prolonged period of time.

(eg, anticoagulants, preservatives, clot activators). When using vacuum tubes containing an additive:

1. Tap the tube gently at a point just below the top to release any additive adhering to the tube or top.
2. Permit the tube to fill completely to ensure the proper ratio of blood to additive. There will be some dead space at the top of the tube.
3. To ensure adequate mixing of blood with the anticoagulant or preservative, use a slow rolling wrist motion to invert the tube gently four to eight times. Failure to invert tubes may lead to the formation of microscopic clots.
4. Rapid wrist motion or vigorous shaking may contribute to hemolysis.
5. Check to see that all the preservative or anticoagulant is dissolved. If any preservative powder is visible, continue inverting the tube slowly until the powder is dissolved.
6. If multiple samples are being drawn, invert each specimen as soon as it is drawn. Do not delay. Place the tube upright in a rack as quickly as possible after collection.

Note: The gel-barrier tube is an additive tube and should be inverted five to six times after collection. Allow the tube to stand for a minimum of 15-30 minutes for complete clotting to occur prior to centrifugation.

When using vacuum tubes containing no additives:

1. Permit the tube to fill completely.
2. Let the specimen stand for a minimum of 15-30 minutes and (preferably) not longer than 60 minutes prior to centrifugation.(NCCLS Guidelines recommends no more than 2 hours.) This allows time for the clot to form. If the specimen is allowed to stand for longer than 60 minutes, chemical activity and degeneration of the cells within the tube will take place, and test results may be affected.
3. Centrifuge the specimen at the end of the waiting period in accordance with the manufacturer's instructions for speed.

Normal serum or plasma is a clear and light yellow to straw in color. Turbid serum or plasma appears cloudy or milky.

Serum or plasma may be cloudy due to bacterial contamination or chronic or transient high lipid levels in the patient's blood.

The primary dietary sources of lipids (fatty substances) are meats, butter, cream, and cheese. Patients who consume these foods within the 24-hour period immediately preceding collection of a blood specimen may have temporarily elevated lipid levels, which may be manifested by cloudy or lipemic serum. Lipemic serum or plasma may not be a true indicator of the patient's physiologic state. Regardless of diet and length of fast, some patients may produce cloudy specimens.

To avoid dietary-induced high lipid levels prior to testing, many physicians require patients to exclude the high-fat foods from their diets or too fast for 12-14 hours prior to specimen collection. For morning specimen collection, the laboratory recommends that the patient be required to fast from 6 PM on the previous evening.

One of the most common problems in specimen collection is the submission of an insufficient volume of specimen for testing. The laboratory sends out a report marked QNS (quantity not sufficient), and the patient has to be called back for a repeat collection at an inconvenience to the patient and to the physician. To ensure an adequate specimen volume:

1. Always draw whole blood in an amount 2½ times the required volume of serum required for a particular test.
2. For example, if 4 mL serum is required, draw at least 10 mL whole blood. If there is difficulty in performing venipuncture, minimum volume may be submitted if it is indicated in the test description. For most profile testing, draw at least two 10 mL gel-barrier tubes. If pediatric tubes are used, be sure to collect an adequate volume of specimen to perform the test.
3. If pediatric tubes are used, be sure to collect an adequate volume of specimen to perform the test.
4. Provide patients with adequate containers and instructions for 24-hour urine and stool collections.
5. It is critical, especially for any specimen collection tube containing an additive, to allow the tube to fill completely with specimen. This requirement is important in order to achieve the proper blood-to-additive ratio; otherwise, the specimen may be found to be QNS.

In general, specimens for determining the concentration of body constituents should be collected when the patient is in a basal state (ie, in the early morning after awakening and about 12-14 hours after the last ingestion of food). Reference intervals are most frequently based on specimens from this collection period.

The composition of blood is altered after meals by nutrients being absorbed into the bloodstream. Consequently, postprandial blood (blood drawn after a meal) is not suitable for some chemistry tests. An overnight fast is preferable (from 6 PM of the evening previous to collection) to ensure that the patient is in the basal state. This minimizes the effects of ingested substances on the test results. Before you collect the specimen, ask the patient when he/she last ate or drank anything. If the patient has eaten recently and the physician wants the test to be performed anyway, you should indicate “nonfasting” on the test request form. In the clinical information/comments section of the test request form, indicate the time the patient ate. Fasting does include abstaining from coffee, tea, or sugar-free products.

Fasting or diet restrictions, such as low-fat diets, should be explained in detail, particularly to aged or overanxious patients or their caregivers. Inform patients that fasting does not include abstaining from water. Dehydration resulting from water abstinence can alter test results.

Fasting or diet restrictions, such as low-fat diets, should be explained in detail, particularly to aged or overanxious patients or their caregivers. Inform patients that fasting does not include abstaining from water. Dehydration resulting from water abstinence can alter test results.

When specimens are not collected in the basal state, the following additional effects should be considered when interpreting test results.

• Exercise. Moderate exercise can cause an increase in blood glucose, lactic acid, serum proteins, and creatine kinase (CK).
• Emotional or Physical Stress. The clinical status of the patient can cause variations in test results.
• Time of Day of Collection. Diurnal variations and variations in circadian rhythm can also affect test results. For example, growth hormone peaks in the morning before waking and decreases throughout the day. Serum iron levels may change as much as 30% to 50%, depending on individual variation, from morning until evening.

Note: For chemistry profiles, 12- to 14-hour fasting specimens are recommended.

Here are some instances in which timed single specimens may be required.

• Fasting plasma glucose alone or in conjunction with a random glucose determination, as recommended by the American Diabetes Association, to diagnose diabetes. Fasting here is defined as no caloric intake for at least 8 hours.
• Postprandial glucose may be performed 2 hours after a meal for a timed test that is helpful in diabetes detection.
• Blood glucose determinations may be ordered at a specific time to check the effect of insulin treatment.
• Blood cultures may be ordered for a specific time if a bloodstream bacterial infection is suspected.
• Therapeutic monitoring of patients on medication.

Here are some instances in which timed multiple specimen tests may be ordered.

• The most common timed procedure is a glucose tolerance test. First, a blood specimen is drawn from a fasting patient. Then, the patient is given glucose orally and blood specimens are drawn at fixed intervals.
  Note: The American Diabetes Association and the World Health Organization (WHO) have specific recommendations for glucose tolerance testing.
• To test the effect of a certain medication, a physician may order the same tests to be obtained on consecutive days, before, during, and after the patient has received a medication.
• Collection of an acute and convalescent serum to aid in the diagnosis of a viral infection when culturing is not feasible.
• Other examples include such tests as occult blood, ova and parasites, and blood cultures.

Serial Monitoring

1. Drugs or their metabolites are frequently concentrated in the urine in sufficient amounts to interfere significantly with urine assays. (See appendices or individual tests for specific information.)
2. Drug interference of notable clinical significance has been well-documented in the following instances.
   • Thiazide diuretic therapy. The pharmacologic or toxic effect is hyperuricemia and hyperglycemia.
   • Catecholamine assay. If a “24-hour drug abstinence period” for a patient is not possible, VMA or metanephrines should be ordered.
   • Oral contraceptives cause a decrease in serum vitamin B₁₂ levels that is, in many cases, indistinguishable from vitamin B₁₂ deficiency of any cause. They also cause an increase in total serum thyroxine-binding globulin. This results in an increase in both total serum thyroxine and unsaturated thyroxine-binding globulin, but with no significant change in unbound (free) thyroxine.
3. Many medications have been shown to have long-term residual effects that interfere with testing.
4. Refer to individual test descriptions for specific information.

Contains no anticoagulant or preservative.

Use: Serum or clotted whole blood. Serum must be separated from cells within 45-60 minutes of venipuncture. Send serum in a plastic transport tube.

Contains clot activator and gel for separating serum from cells, but not anticoagulant. Do not use gel-barrier tubes to submit specimens for therapeutic drug monitoring. Always check the test description to determine whether a gel-barrier tube is acceptable.

Use: Serum. May be used for assays requiring serum unless otherwise stated. Separate serum from cells within 15-30 minutes of venipuncture. Serum may be sent in the centrifuge tube with an intact barrier (correct separation upon centrifugation) between cells and serum or in a plastic transport tube. If specimen is centrifuged before clotting is complete, a fibrin clot will form on top of the cell. This finding is frequent in hemolyzed specimens. Also, the gel barrier may not be intact and could cause improper separation of serum and cells, possibly affecting test results.

Contains K₂ EDTA.

Use: EDTA whole blood or plasma. Send plasma in a plastic transport tube labeled “Plasma, EDTA.” Send whole blood in a lavender-top tube.

Contains sodium fluoride (a preservative) and potassium oxalate (an anticoagulant).

Use: Sodium fluoride whole blood or plasma. Send plasma in a plastic transport tube labeled “Plasma, Sodium Fluoride.” Send whole blood in a gray-top tube.

Contains sodium citrate. Be sure to use only tubes with a 3.2% sodium citrate concentration. These are easily identified by the yellow diagonal stripes on the label.

Use: Sodium citrate plasma. Send plasma in a plastic transport tube labeled “Plasma, Sodium Citrate.” Send whole blood in a blue-top tube.

Contains sodium heparin or lithium heparin.

Use: Heparinized whole blood or plasma. Send plasma in a plastic transport tube labeled “Plasma, Sodium Heparin” or “Plasma, Lithium Heparin.” Send whole blood in a green-top tube.

Contains acid citrate dextrose (ACD) solution.

Use: ACD whole blood. Send whole blood in a yellow-top tube.

Contains sodium EDTA for trace metal studies.

Use: EDTA whole blood or plasma. Send whole blood in a royal blue-top tube. Send plasma in a plastic transport tube labeled “Plasma, EDTA from royal blue”.

Contains EDTA.

Use: EDTA plasma for molecular diagnostic tests (eg, polymerase chain reaction (PCR) and/or branched DNA amplification (bDNA) techniques). Upon centrifugation, a gel barrier is formed between the plasma and the cellular components of the blood. The tube can be sent directly to the lab without transferring to a secondary tube. Plastic tubes can be frozen at -80°C without risk of breakage.

Contains EDTA.

Although plastic red-top tubes and gel-barrier tubes are becoming the standard for assays performed on serum, they are not preferred in all situations. Plastic tubes are not appropriate for blood drug screens or serum or blood alcohol tests. The use of plastic tubes may yield inaccurate results; consequently, the preferred container for these assays is glass. Specimens for the following procedures should not be submitted in plastic tubes.

• Cannabinoids (Marijuana), Blood  
• Drug Coma/Overdose Profile II, Blood and Urine or Gastric Contents
• Drug Coma/Overdose Profile II, Blood and Urine (With Cannabinoids)
• Drug Profile, Blood (5 Drugs)
• Drug Profile, Blood (7 Drugs)
• Ethanol, Blood
• Methanol, Blood
• Volatiles, Blood
• Zinc, Plasma/Serum

Contains EDTA.

Assemble the following supplies: lab coat, gloves, labels, safety needle, needle holder, tourniquet, appropriate tubes, gauze, alcohol sponge, adhesive strip, and sharps container. Put on the lab coat and gloves. The aseptic method of collecting and transporting a blood specimen works on the principle of a vacuum tube for drawing blood. A double-pointed needle or multiple sample 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 1½ inches permits an angle of entry that will not pierce both vein walls and enter tissue.

When more than one blood specimen is required, multiple sample needles and vacuum tubes make blood collection simpler and more efficient. 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.

Place the sharps container within reach. Open the single or multiple sample needle packages in front of the patient; do not tear the paper seal on the needle and remove the needle sheath (sterile shield).

Prepare the needle holder in order to attach the safety needle in the appropriate manner. Pull the safety shield on the needle back over the holder before removing the needle shield. Thread the needle into the holder and tighten it firmly. Follow the manufacturer's recommendations on properly setting the needle. With some needle assemblies, you may slide the collection tube into the holder, carefully pushing the tubes forward until the needle touches the stopper. 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.

During venipuncture, do not have the patient clench and unclench the fist repeatedly (“pumping”). This will cause a shift in fluid between the vein and the surrounding tissue. This can lead to changes in concentration of certain analytes. To facilitate making the vein more prominent, the patient may be asked to hold firmly to a rubber ball, a thick wad of gauze, etc. 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.

After securing the tourniquet and reaffirming your selection of the best vein, both by sight and palpation, proceed as follows. Note: If a patient has intravenous (I.V.) solutions going into one or both arms, it is acceptable to puncture the vein 3-4 inches below the site of the I.V.

1. Except when a 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. Do not routinely use an iodine preparation. Iodine may contaminate specimens for certain chemistry tests.
2. Allow the puncture site to air dry after swabbing, or dry the site with gauze. If alcohol is not allowed to dry, it may cause specimen hemolysis. If the arm is dry, you will avoid stinging the patient at venipuncture.
3. Break the paper seal on the needle cap and remove the needle cap. Visually inspect the point of the needle for burrs and possible discoloration along the shaft of the needle before using the needle. If it has burrs or discoloration, do not use that needle; use another fresh, sterile, unused needle.
4. Anchor the vein. Enter the vein with the needle at an angle of approximately 15-20 degrees.

If only a single collection tube is required, when the vacuum is exhausted and the tube completely filled, releases the tourniquet, and remove the tube from the needle assembly. Place a piece of dry gauze over the needle and withdraw the needle carefully.

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 and invert each successive tube after it is filled. When all samples have been drawn, remove the entire assembly from the arm. Firmly lock the safety shield on the needle. Dispose of the used needle according to the provisions in your exposure control plan. Do not recap, cut, or bend any needles; dispose of them in a safe, responsible manner. Do not reuse needles.

Apply direct pressure to the puncture site. After drawing blood, observe proper venipuncture techniques to prevent continued bleeding and/or hematoma. Excessive bleeding (longer than 5 minutes) should be brought to the attention of the physician. Also, a clot tube (eg, red-top tube or gel-barrier) that does not clot should be brought to the attention of the physician.

Note: When multiple specimens are drawn from a single venipuncture, the following order is recommended: (1) sterile blood culture tubes, (2) nonadditive clotting tubes (red), (3) coagulation tubes and tubes containing citrate (blue), (4) gel-barrier tubes and tubes with additives (red), (5) tubes containing heparin (green), (6) tubes containing EDTA (lavender, royal blue), (7) tubes containing acid citrate dextrose (yellow), and (8) tubes containing sodium fluoride and potassium oxalate (gray).

Note: If the blood has to be mixed with an additive (invert the tube 4-8 times), this must be done immediately after collection. 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. As soon as possible after collection, set the blood upright in a test tube rack.

• Label tubes in front of the patient immediately after collection, confirming all necessary information with the patient.
• If blood is drawn for routine hematology, prepare the blood films (blood smears) immediately after collection.
• Complete the test request form to indicate time and date of collection along with collector's identification.

Note: For pediatric patients and some geriatric patients, the total volume of blood collected should be noted on the test request form. Additional steps may be required in some locations.

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

1. Draw whole blood in an amount 2½ 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. 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 30-60 minutes. If clotting fails to occur within 60 minutes, notify the physician. Do not remove the tube stopper.
3. After allowing clot to form, insert the tube in the centrifuge, stopper end up. Operate the centrifuge for no more than 10 minutes at the speed recommended by the manufacturer. Prolonged centrifugation may cause hemolysis. When using a bench-top centrifuge, employ a balance tube of the same type containing an equivalent volume of water.
4. Turn the centrifuge off, if not automatic turn off, and allow it to come to a complete stop. Do not attempt to open the lid and stop by hand or brake. Remove the tube carefully without disturbing the contents. Do not spin more than 10 minutes unless otherwise specified.
5. Remove the stopper and carefully aspirate all serum from cells, using a separate disposable pipette for each tube.
6. Place the tip of the pipette against the side of the tube, approximately ¼ inch above the cell layer. Do not disturb the cell layer or carry any cells over into the pipette. If cells do enter the pipette, re-centrifuge the entire specimen.
7. Transfer the serum from the pipette into the transport tube. 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.
8. 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.

When frozen serum is required, place the plastic transport tube(s) immediately in the freezer compartment of the refrigerator. Notify your professional service representative that you have a frozen specimen to be picked up. A separate frozen sample must be submitted for each test requiring a frozen specimen. Never freeze the original tube. Glass tubes break when frozen and create a safety hazard.

Note: Freeze (frozen) is defined as causing a specimen to pass from liquid to solid by loss of heat. A frozen specimen should be held in a freezer at 0°C to -20°C unless a specific test requires the specimen to be frozen at -70°C (dry ice).

1. If you have after-hours pickup for frozen specimens, place an ice pack in the freezer so that it may freeze. Separate the specimen, either plasma or serum, from the cells, and place the required amount of specimen into the special transport tube. Label the tube with a permanent marker. (Water-soluble markers may wash off with freezing and transport.) Place the tube(s) in a designated freezer. Just prior to leaving the office, place the frozen transport tube together with an ice pack in the specimen bag and seal. Ice packs can keep frozen specimens frozen, but they will not be able to freeze ambient specimens.
2. Put the specimen bag containing the specimen with the ice pack in your lockbox. Your professional services representative will remove the transfer tube from the specimen bag and put it on dry ice. The ice pack will be left in your lockbox for reuse. If a specimen is collected just prior to closing and does not have time to freeze prior to pickup, place the specimen with the frozen ice pack, and leave a note that the specimen has not had time to freeze so that the professional service representative will place the specimen on dry ice. Specimens for multiple tests should be frozen in different transfer tubes.

Gel-barrier (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 gel-barrier tube. Do not use gel-barrier tubes to submit specimens for therapeutic drug monitoring, direct Coombs', blood group, and blood types. There are other times when gel-barrier tubes should not be used. Always consult the test description prior to collection.

1. Draw whole blood in an amount 2½ 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. Label the specimen appropriately.
2. Gently invert the gel-barrier tube five times to mix the clot activator and blood.
3. Place the collection tube in the upright position in the rack, and allow the blood to clot at room temperature for no longer than 60 minutes. (Clots usually form in 15-30 minutes.)
4. After allowing the clot to form, insert the tube in the centrifuge, stopper end up. Operate the centrifuge for 10 minutes at the speed recommended by the manufacturer. Prolonged centrifugation may cause hemolysis. When using a bench-top centrifuge, employ a balance tube of the same type containing an equivalent volume of water. Do not exceed 10 minutes of spin time unless otherwise specified.
5. Turn the centrifuge off, if not an automatic turn 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. Inspect the barrier gel to ensure that it has formed a solid seal between the serum and 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 transport tube. Unless otherwise indicated, serum specimens may be sent at room temperature.
8. When frozen serum is required, follow the steps in number 9 above.

When plasma is required, follow these steps.

1. Always use the proper vacuum tube for tests requiring a 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.
3. Permit the vacuum tube to fill completely. Failure to fill the tube will cause an improper blood-to-anticoagulant ratio and yield questionable and/or QNS test results.
4. To avoid clotting, mix the blood with the anticoagulant or preservative immediately after drawing each sample.
5. To ensure adequate mixing, slowly invert the tube eight to ten times (four times for citrate tubes) using a gentle wrist rotation motion.
6. Immediately centrifuge the specimen for 10-15 minutes or as specified by the tube manufacturer. Do not remove the stopper.
7. Turn the centrifuge off, if not an automatic turn 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.
8. Remove the stopper and carefully aspirate plasma, using a separate disposable Pasteur pipette for each tube.
9. Place the tip of the pipette against the side of the tube, approximately ¼ inch above the cell layer. Do not disturb the cell layer or carry any cells over into the pipette. Do not pour off; use transfer pipette.
10. Transfer the plasma from the pipette into the transport tube. Be sure to provide the laboratory with the amount of plasma specified.
11. 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 (eg, “Plasma, Sodium Citrate,” “Plasma, EDTA,” etc).
12. 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.
13. Never freeze glass tubes. For after-hours pickup, follow the steps under number 9 under Preparing Serum above.

The BD Vacutainer® Plasma Preparation Tube (PPT™) is a plastic evacuated tube used for the collection of venous blood in order to prepare undiluted plasma for use in molecular diagnostic testing.

1. The BD PPT™ should be at room temperature and properly labeled for patient identification.
2. Collect blood into the BD PPT™ following standard procedure for venipuncture and sample collection. Permit the vacuum tube to fill completely. Failure to fill the tube will cause an improper blood-to-anticoagulant ratio and may yield questionable and/or QNS test results.
3. To avoid clotting, mix the blood with the anticoagulant immediately after drawing each sample.
4. To ensure adequate mixing, gently invert the BD PPT™ eight to ten times using a gentle wrist rotation motion.
5. After mixing, store the BD PPT™ upright at room temperature until centrifugation. Blood samples should be centrifuged within 2 hours of blood collection for best results. Centrifuge BD PPT™/blood sample at room temperature at 1100 RCF (relative centrifugal force) for a minimum of 10 minutes in a swinging bucket rotor type centrifuge. (Use of a fixed angle rotor centrifuge does not allow the gel barrier to form properly and may result in incomplete separation of plasma from the cellular components.)
6. Allow centrifuge to come to a complete stop before attempting to remove tubes. Examine tube to ensure that the gel barrier has formed between the plasma and the cellular elements.
7. Once centrifuged, the plasma in the BD PPT™ can be transported to the lab without transferring to another tube. The gel barrier prevents the remixing of the plasma with the cellular elements of the blood. The plastic BD PPT™ can be frozen at -80°C prior to shipment.

The blood film (commonly called a blood smear) can be a vital part of clinical testing. When employed, it enables the technologist to view the actual physical appearance of the red and white blood cells microscopically.

Well-prepared films can be used in performing the differential white cell count, for examining the morphology (size, structure, and shape) of red and white cells to determine the presence of abnormal cells, and also for the examination of the size and number of platelets. The distribution of the cells, as well as their morphology, can be altered by poor slide preparation.

The most appropriate slide consists of a film that is exactly one cell thick for maximum visualization of all cell types microscopically.

Blood films may be prepared from venous blood (venipuncture) or capillary puncture blood. Slide preparation using venous blood is described below.

Follow the steps outlined below.

1. Put on lab personal protective equipment.
2. Select two clean, grease-free glass collection slides with frosted ends (new ones whenever possible).
3. Print the patient's name and date on the frosted ends of both slides. (See Figure 15.)
4. Handle all slides only by the frosted ends or by the edges.
5. Place the collection slides frosted side up and to your right on a padded, flat surface near the chair or bed where the specimen is to be collected.
6. Immediately after removing the needle from the vein, gently touch the tip of the needle to one of the clean slides, producing a small drop of blood about 1-2 mm in diameter, about the size of a match head. The drop of blood should be in the center line, approximately ¼ inch from the frosted end. Repeat for the second collection slide. Activate the needle's safety feature and dispose of the needle in a sharps container.
7. Hold the left corners of the collection slide with the left thumb and forefinger.
8. Hold the spreader by the frosted end between the right thumb and the index finger.
9. Rest the left end of the spreader at a 45° angle, approximately ½ inch opposite the drop of blood on the slide. This angle prevents the white cells from bunching along the edges.
10. Draw the spreader slide steadily back toward the drop of blood. When the slide contacts the drop, the blood will start to spread to the edges of the spreader slide. (See Figure 16.)
11. Keep the spreader slide at a 45° angle, maintaining light but firm pressure with the spreader slide against the horizontal slide. Push the spreader slide rapidly over the entire length of the slide, pulling a thin smear of blood behind it. A feathered edge usually characterizes a good blood film. (See Figure 17.)
12. Prepare the second film in the same manner.
13. Allow the blood films to air dry. Do not blow on the slides. Do not apply fixative. After the slides are completely dry, place them in a labeled slide holder for transport to the laboratory.

Here are some instances in which timed single specimens may be required.

1. Slides must not be touched on any area except the long slide edges or frosted ends.
2. Prepare the film immediately, as soon as the drop of blood has been placed on the slide. Any delay will result in abnormal distribution of the white cells, with many of the larger white cells accumulating at the thin edge of the smear. Rouleaux of the red cells (stacking like piles of coins) and platelet clumping will also occur.
3. Criteria of a good blood film.
   • The thin portion should be about 1 inch long, and the entire film should cover approximately half of the area of the entire slide.
   • No portion of the film should extend to the edges of the slide.
   • The film should be free of waves, holes, and ridges, and it should have a smooth appearance and feathered edge.
   • Label the frosted area of the slide with the appropriate information.
4. Common causes of a poor blood film. (See Figure 18.)
   • Too long a delay in transferring the drop of fresh blood from collection tube to slide.
   • Drop of blood too large or too small (usually too large).
   • Spreader slide pushed across the slide in a jerky manner.
   • Greasy or dirty slides, or use of a spreader slide with a chipped or unpolished end.
   • Failure to keep the entire edge of the spreader slide against the slide while making the film.
   • Failure to keep or have the spreader slide at approximately a 45° angle. (Increasing the angle results in a thick film, while a smaller angle will produce a thin film.)
   • Failure to push the spreader slide completely across the flat slide.

Laboratory tests requiring urine specimens involve a wide variety of procedures. A basic urinalysis is almost always included in the routine work-up of patients. When a urine culture or a more esoteric urine test is ordered, the clinical usefulness of the test results can be assured only if the patient receives explicit written instructions.

Submit a urinalysis preservative tube and culture and susceptibility preservative tube. Label both filled tubes with patient's name and date and time of specimen collection.

Submit a urinalysis preservative tube. Label filled tube with patient's name and date and time of specimen collection.

Submit a culture and susceptibility preservative tube. Label the filled tube with patient's name and date and time of specimen collection.

1. The collection of the 24-hour urine starts with the patient voiding (completely emptying bladder) and discarding the first urine passed in the morning.
2. Except for this first discarded urine, all of the urine passed during that day and night, up to and including the first voiding of the following day, must be collected. Urine passed during bowel movements must also be collected.
3. If possible, the entire specimen should be refrigerated at 2°C to 8°C during collection, or kept in a cool place, since urine is an excellent culture medium for organisms, and its components decompose quickly.
4. The 24-hour urine container may contain a preservative of acetic acid, boric acid, or hydrochloric acid, which may cause burns if touched. If ingested, a physician should be contacted immediately.

1. A normal intake of fluids during the collection period is desirable unless otherwise indicated by the physician or test specimen requirements.
2. In some cases, it may be advisable for patients to discontinue taking all medications for an interval of at least 12 hours (preferably 48-72 hours) preceding the urine collection period. This is done as a precaution against interference in the chemical assays of various hormones; there may be instances, however, in which this is not recommended.
3. In certain complex chemical analyses, the metabolic products of certain foods may also cause misleading results. In these instances, the laboratory will advise the physician of special dietary restrictions to be communicated to the patient.

1. If only a portion of the 24-hour collection is submitted to the laboratory, be sure to measure the entire 24-hour volume and record the total amount in milliliters (mL) on the test request form for laboratory use. The specimen must be well mixed prior to pouring off aliquots of the sample into an appropriately labeled transport container and submitting it to the laboratory.
2. When aliquots are required with preservatives, use only the preservative requested. One preservative must not be substituted for another. Do not add preservatives except as specified by the laboratory.
3. Even in the healthy individual, the total daily urine volume may be highly variable depending upon water intake, diet, activity, and environmental factors. Be sure to provide the patient with a container of adequate size.
4. When the laboratory requires a pooled, well-mixed specimen (collected in more than one container) or a measured aliquot for an individual test, the specimen must be prepared exactly as stated in the specimen requirement. Submit this to the laboratory as soon as possible.

This section includes written instructions to be provided to the patient with the specified laboratory collection container. Supplement these instructions by discussing them with the patient and explaining why the test and collection procedures are necessary. Collection containers that include acids should be clearly marked.

1. Upon rising in the morning, urinate into the toilet, emptying your bladder completely. Do not collect this sample. Note the exact time and print it on the container label.
2. Collect all urine voided for 24 hours after this time in the container provided by the physician. All urine passed during the 24-hour time period (day or night) must be saved. Urine passed during bowel movements must also be collected.
3. Refrigerate the collected urine between all voiding or keep it in a cool place.
4. At exactly the same time the following morning, void completely again (first time after awakening), and add this sample to the collection container. This completes your 24-hour collection.
5. Take the 24-hour specimen to the physician's office or laboratory as soon as possible, maintaining the cool temperature in transit by placing the specimen in a portable cooler or insulated bag.

1. Provide the patient with two 24-hour specimen containers, one containing preservative (30 mL hydrochloric acid) and one containing no preservative.
2. This 24-hour urine container contains hydrochloric acid, which may cause burns if touched. If ingested, a physician should be contacted immediately.
3. Have the patient collect two consecutive 24-hour urine specimens according to the collection protocol provided in the accompanying patient directions. The first 24-hour specimen should be collected in the container with preservative. The specimens must be refrigerated during collection, and the two specimens should be returned to the office/laboratory as soon as possible after completion of the second collection.
4. Upon return of the two 24-hour urine specimens, they should be handled as follows.
   1. Measure the total volume of each 24-hour specimen and record these volumes (in mL) on the test request form under “Clinical Information.” Be sure to identify which specimen was collected with preservative and which was collected without preservative. Example:

2. After measuring the volumes, pour off an aliquot from each specimen into the special urine containers provided. These containers should be filled approximately three-fourths full (about 75 mL).
3. Label each aliquot with the patient's full name, the measured 24-hour urine volume, and whether the specimen was collected with or without preservative. Special kidney stone prevention profile labels are provided for your convenience.
4. Upon return of the 24-hour urine specimens, a fresh random urine specimen should be obtained from the patient for pH determination. Measure the pH urine using a dipstick, and record the value on the aliquot labels as well as on the test request form under “Clinical Information” (below the two urine volumes). This specimen should then be discarded.
5. Complete the test request form by providing all necessary information.
6. Complete the patient history (questionnaire), including all information required, and mail this to the laboratory. It is mandatory that this history be submitted; otherwise, a physician interpretation will not be performed. A prepaid envelope is supplied for your convenience.
7. Place the two urine aliquots in the plastic zip-lock specimen bag labeled “Kidney Stone Prevention.” The completed test request form goes in the side pocket of the bag. Store in the freezer.

To the Physician: You may wish to photocopy these pages so that you can provide your patients with written instructions.

1. Urinate in the usual manner on awakening, making sure to empty your bladder completely. Do not save this urine, but you must record the date and time of this first urination. Example: 03/13/01, 7:30 AM.
2. All urine passed during the remaining 24-hour period must be collected in this first container, labeled “HCl Preservative.” Urine passed during bowel movements must also be collected.
3. Urine may be collected in another clean container and then carefully poured into the first 24-hour collection container.
4. The next morning, urinate on awakening, but this time includes the urine in the HCl preservative container. Record the date and time of this urination. Example: 03/14/03, 7:30 AM. This is the last sample to be included in the container marked “HCl Preservative.”

1. Record the date and time of the first urine of the day. The time is the same as the last entry of the HCl preservative container. (See number 4 above.)
2. From now on, all urine passed for the next 24-hour period must be included in the second container, labeled “No Preservative.” Urine passed during bowel movements must also be collected.
3. Urine may be collected in another clean container and then carefully poured into the second container.
4. On the following morning, the first urine of the day must be included in this second container. Record the date and time of this urination. Example: 03/15/03, 7:30 AM. This is the last sample to be included in the container labeled “No Preservative.”

Here are some instances in which timed single specimens may be required.

1. Collection Tube. Blood should be collected in a blue-top tube containing 3.2% buffered sodium citrate.
2. High Hematocrit Samples. Patients with elevated hematocrits have a relatively low amount of plasma for a given whole blood (collection) volume. This tends to effectively increase the plasma citrate concentration. If the patient has a known hematocrit >55%, the amount of citrate in the collection tube must be decreased according to the formula below:

3. Example:
4. Patient hematocrit = 60%
   Total volume = 5 mL (standard citrated plasma collection tube volume)

5. Order of Draw. A discard tube is not required prior to collection of coagulation samples. When noncitrate tubes are collected for other tests, collect sterile and no additive (red top) tubes prior to citrate (blue top) tubes. Any tube containing an alternate anticoagulant should be collected after the blue-top tube. Gel-barrier tubes and serum tubes with clot initiators should also be collected after the citrate tubes.
6. Venipuncture Technique. To avoid contaminating the sample with tissue thromboplastin, the venipuncture must be clean, with no trauma. Hemolyzed samples are not acceptable.
7. Winged blood collection kits (butterfly) must use a discard lead tube prior to collecting specimen tube to submit for testing.
8. Fill Volume. Evacuated collection tubes must be filled to completion to ensure that a 9:1 blood-to-anticoagulant ratio is achieved. Underfilling of citrate collection tubes results in an increased anticoagulant-to-blood ratio and can extend clot-based coagulation assays. Note: Never combine two undefiled tubes together.
9. Mixing. The sample should be mixed immediately by gentle inversion at least six times to ensure adequate mixing of the anticoagulant with the blood.
10. Lupus anticoagulants (LA) are nonspecific antibodies that extend clot-based coagulation assays as the result of their interaction with phospholipids in the reaction mixture. Platelets in plasma samples can act as a source of phospholipids and mask the effects of LA. For this reason, it is important to prepare platelet-poor plasma (PPP) for LA testing. PPP should have a platelet count <10,000/mcL. PPP samples should be collected by double centrifugation.

11. Centrifuge for 10 minutes and carefully remove two-thirds of the plasma using a plastic transfer pipette, being careful not to disturb the cells.
12. Deliver plasma to a plastic transfer tube, cap, and recentrifuge for 10 minutes.
13. Use a second plastic pipette to remove the plasma, staying clear of the platelets at the bottom of the tube.
14. The specimen should be frozen immediately and maintained frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.

Lenco test offerings are based on the 2001 Consensus Guidelines for the Management of Women With Cervical Cytological Abnormalities, reported in JAMA on April 24, 2002 (JAMA, 287(16):2120-8. Lenco’s Thin Layer Prep Pap Test with Reflex to Only High-Risk HPV Hybrid Capture When ASCUS will reflex to testing with high-risk HPV probes only when the Pap result category is ASC-US. This is the approach recommended in the 2001 Consensus Guidelines.

Federal regulations require that all specimens be accurately identified. Consequently, the patient's name must be clearly written on the frosted end of the slide or on the liquid-based collection container. Unidentified cases will be returned to your office unprocessed. Please note the patient's date of birth and Social Security number on the test request form. This patient information will aid in compiling and maintaining a 5-year database for retrieval of previous cytology reports.

Note: In accordance with criteria established by CLIA, Pap smears will be referred for pathologist review if laboratory personnel suspect (1) reactive or reparative cellular changes, (2) atypical squamous or glandular cells of undetermined significance, or (3) cells in the premalignant or malignant category. In these cases, additional CPT code(s)/charge(s) may apply. (Slides that are routinely reviewed by a pathologist for quality control purposes are not included.)

• Indicate conventional smear (Pap smear) vs liquid-based vs other.

• Satisfactory for evaluation (describe presence or absence of endocervical/transformation zone component and any other quality indicators, eg, partially obscuring blood, inflammation, etc).
• Unsatisfactory for evaluation (specify reason).
• Specimen rejected/not processed (specify reason)
• Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of (specify source).

• Negative for intraepithelial lesion or malignancy
• Epithelial cell abnormality: See Interpretation/Result (specify “squamous” or “glandular” as appropriate).
• Other: See Interpretation/Result (eg, endometrial cells in a woman 40 years of age).

• Provide a brief description of the test methods and report the result so that it is easily understood by the clinician.

• Negative for intraepithelial lesion or malignancy (when there is no cellular evidence of neoplasia, state this in the Interpretation/Result section of the report, whether or not there are organisms or other non-neoplastic findings.

• Trichomonas vaginalis
• Fungal organisms morphologically consistent with Candida spp
• Shift in flora suggestive of bacterial vaginosis
• Bacteria morphologically consistent with Actinomyces spp
• Cellular changes consistent with herpes simplex virus

• Reactive cellular changes associated with inflammation (includes typical repair)
• Radiation
• Intrauterine contraceptive device (IUD)
• Glandular cells status posthysterectomy
• Atrophy

• Endometrial cells (in a woman 40 years of age). Specify if “negative for squamous intraepithelial lesion.”

• Atypical squamous cells of undetermined significance (ASC-US)
• Atypical squamous cells of undetermined significance, cannot exclude HSIL (ASC-H)
• Low-grade squamous intraepithelial lesion (LSIL) – encompassing: HPV / mild dysplasia / CIN
• High-grade squamous intraepithelial lesion (HSIL) – encompassing: moderate and severe dysplasia, CIS/CIN 2 and CIN 3 with features suspicious for invasion (if invasion is suspected)
• Squamous cell carcinoma

• Atypical endocervical cells (not otherwise specified NOS; or specify in comments)
• Atypical endometrial cells (NOS or specify in comments)
• Atypical glandular cells (NOS or specify in comments)
• Atypical endocervical cells, favor neoplastic
• Atypical glandular cells, favor neoplastic
• Endocervical adenocarcinoma in situ
• Adenocarcinoma endocervical
• Adenocarcinoma endometrial
• Adenocarcinoma extrauterine
• Adenocarcinoma NOS

• Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included).

1. Submit blood or bone marrow in sterile sodium heparin (green) tubes. Lithium heparin tubes (green) inhibit cell growth. Use a small pediatric tube for aspirates less than 4 mL in volume. This will help avoid heparin toxicity of the sample.
2. Submit fetal tissue, skin, or other biopsies in Ringer's lactate, Hanks' Balanced Salt Solution, or Lenco transport medium.
3. Only refrigerate specimens when sterility is questioned.

1. Submit specimens in sterile plastic tubes. To discard, cap the tip of the syringe and secure the plunger with tape.
2. Maintain at ambient temperature. Do not freeze or refrigerate.
3. Do not use the following containers for amniotic fluid:
   1. Glass tubes with rubber stoppers. Rubber is toxic to amniocytes.
   2. Wide-mouth urine containers those are prone to leak and become contaminated.
4. When preparing to transport cytogenetics specimens, please follow these guidelines.
   1. Ship all specimens so they reach the laboratory by midweek and within 48 hours of collection.
   2. To facilitate turnaround time and communications between the laboratory and physicians, complete the chromosome analysis history and order form with all the information required and staple the appropriate test request form to it. The following items are especially important:
      • Name and telephone number of the attending physician, which allows us to call abnormal results. (Written reports follow at the regularly scheduled times.)
      • Clinical data, to help interpret ambiguous karyotypes or determine the need for additional studies (eg, specialized banding techniques, extra chromosome counts).
      • Be sure that both the sample submitted and the tests requested are correctly indicated.
      • Please note that you have the option to order a quantitative alpha-fetoprotein test on the same specimen for an additional charge. The minimal gestational age for amniotic fluid alpha-fetoprotein is 12 weeks.

1. Remove 0.5-1.0 g of solid tumor tissue, trim away excess fat and blood, and cut tissue into small pieces, then quick-freeze the specimen on dry ice, in a cryostat, or in liquid nitrogen within 20 minutes of excision. Do not wrap in gauze or aluminum foil. Store at -70°C or on dry ice.
2. Place the tissue pieces in a 60 mL biopsy bottle (fluorescent pink) without formalin. Be sure the bottle cap is tightly secured. Prior to freezing, use a waterproof marker to label the bottle with the patient's name, test requested, and your account number. Tape or gummed labels may not adhere to the bottle at -70°C.
3. Seal the bottle in one pouch of a two-pouch specimen bag. Place the test request form in the second pouch. As an extra precaution, tape the top of the test request form pouch to ensure that the test request form remains with the specimen.
4. If multiple specimens from a single patient are submitted, each specimen should be placed in a separate bottle and sealed in a two-pouch specimen bag with its own test request form. Be sure each bottle and test request form is labeled with the patient's name, tissue site, and your account number.

1. Remove 0.5-1.0 g of solid tumor tissue, trim away excess fat and blood, and cut tissue into small pieces. Quick-freeze the specimen on dry ice, in a cryostat, or in liquid nitrogen within 20 minutes of excision. Do not wrap in gauze or aluminum foil. Store at -70°C or on dry ice.
2. Place the tissue pieces in a 60 mL biopsy bottle (fluorescent pink) without formalin. Be sure the bottle cap is tightly secured. Prior to freezing, use a waterproof marker to label the bottle with the patient's name, test requested, and your account number. Tape or gummed labels may not adhere to the bottle at -70°C.
3. Seal the bottle and the completed test request form in a plastic zip-lock bag. As an extra precaution, staple the top of the bag to ensure that the test request form remains with the specimen.
4. If multiple specimens from a single patient are submitted, each specimen should be placed in a separate bottle and sealed in a plastic two-pouch specimen bag with its own test request form. Be sure each bottle and test request form is labeled with the patient's name, tissue site, and your account number.

1. Centrifuge fluids to obtain a cell pellet. The minimum acceptable specimen volume is 0.2 mL packed cells.
2. Wash the cell pellet with a physiological saline or similar isotonic nonprotein, nonpreservative medium. A single 15- or 50 mL plastic centrifuge tube with a screw cap should be used for this process.
3. Drain liquid from the cell pellet. Use a waterproof marker to label the tube with the patient's name, test requested, your account number, and the words “Effusion Cell Pellet.”
4. Be sure the tube cap is tightly secured, and then quick-freeze the specimen on dry ice, in a cryostat, or in liquid nitrogen.
5. Seal the tube and the completed test request form in a plastic zip-lock bag.

• Expired collection device
• Inappropriate collection device/specimen type
• Unlabeled specimens
• Mislabeled specimens (patient ID not corresponding to test order)
• Leaking/broken specimen container
• Gross bacterial contamination of specimen
• Quantity of specimen not sufficient for test
• Specimen subjected to extensive delay or extreme temperatures

• Acetic acid (30%) solution
• Boric acid tablet
• Citrate buffered acetone solution
• Ethyl alcohol (ethanol) solution
• Ethyl alcohol and polyethylene glycol solution
• Formalin (formaldehyde) – 10% neutral buffered formalin solution
• Hydrochloric acid solution
• Isopropyl alcohol solution
• Methyl alcohol (methanol) solution
• Perchloric acid (8%) solution