General Specimen Collection

Phlebotomy
General Blood Collection
Blood Collection by Type
Common Causes for Unacceptable Blood Specimens and Inaccurate Test Results
Frozen Specimens
Random Urine Collection
24 Hour Urine Collection
Urine Preservative Chart
Timed Stool Collection
Coagulation Sample Collection

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Phlebotomy

Most blood specimens can be obtained using routine phlebotomy techniques; however, there are some exceptions. The use of a tourniquet can cause stress and is not recommended in some cases. Patients should be instructed not to clench their fist(s) prior to or during the phlebotomy procedure as this may alter some of the patient’s laboratory results, such as the concentration of potassium in serum. The patient’s posture (sitting, standing or supine), or the time of day of phlebotomy can be important factors for some tests (eg, therapeutic drug monitoring and hormone tests). Special collection instructions are associated with those tests that require them in the Test Directory.

General Blood Collection

Draw blood in the appropriate color-coded Vacuette® tube (see chart below). For serum or plasma, draw approximately 2 1/2 times the requested volume. For serum, allow the blood to clot 30-60 minutes and then separate by centrifugation. For plasma and whole blood, completely fill the vacutainer whenever possible to eliminate dilution from the anticoagulant or preservative and immediately mix the blood by gently and thoroughly inverting the tube five to ten (5-10) times. Separate plasma by centrifugation. Transfer the serum, plasma, or whole blood to a plastic transport tube. To prevent injury and exposure to potentially infectious material, do not ship frozen serum, plasma, or whole blood received in glass tubes or SST (glass or plastic).

The color-coded vacutainer tubes are recommended unless otherwise indicated in the Test Directory. Upon request, color-coded pediatric vacutainer tubes can be provided to facilitate special handling.

Vacuette^® Collection Tube Guide
Courtesy of greiner bio-one

Blood Collection by Type

Whole Blood

Collect whole blood according to instructions provided for the individual test. Thoroughly mix the blood with the additives by gently inverting the tube according to the instructions in chart above (4 times in the case of light blue-top (sodium citrate) tubes). Maintain the specimen at room temperature or on cool packs before submitting to the laboratory unless instructed otherwise by the specimen requirements. Never freeze whole blood unless specifically instructed in the specimen requirements.

For cool packs stored in the freezer, be sure to allow enough time for them to warm to refrigerator temperature before placing whole blood specimens near them. To minimize the risk of hemolysis, do not place whole blood specimens in direct contact with cool packs.

Serum

Please check individual specimen requirements for restrictions. When using a serum separator tube, follow these instructions:

Perform venipuncture as with any other blood collection device.
Invert the tube gently no more than ten (10) times. Further inversion may cause alterations in sample integrity.
Do not remove the stopper at any time. Do not centrifuge immediately after drawing blood. Allow the blood to clot in an upright position for at least 30 minutes but not longer than one (1) hour before centrifugation.
Centrifuge for 10-15 minutes at 1600-2200 RPM* within one (1) hour of collection. Quest recommends swing-out centrifuges and higher speed (g) for better cell separation.
Transfer the serum to a plastic screw-cap vial for transport to the laboratory.

Note: Do not use serum separator tubes for therapeutic drug monitoring or toxicological analysis. The plastic serum separator material extracts lipophilic substances (most drugs), resulting in a falsely low drug concentration result. Instead, collect the specimen in a plain red-top tube containing no anticoagulants or preservatives. Transfer the serum with a pipette to a plastic vial for transport to the laboratory. Serum should be clear and free from all red cells.

Plasma

Plasma contains fibrinogen and other clotting factors when separated from the red blood cells. Evacuated tubes used to collect plasma specimens contain anticoagulant and, frequently, a preservative. The additive in each tube is specified on the label and tube stoppers are color coded according to the additive present. Consult the individual test specimen requirements to determine the correct additive/tube to use. Indicate that the specimen is plasma on the plastic screw-cap vial for transport and test requisition.

When plasma is required, follow these instructions:

Draw 12 mL of whole blood for each 5 mL of plasma needed. Collect in an appropriate collection tube.
Centrifuge for 10-15 minutes at 1600-2200 RPM* within one (1) hour of collection. Quest recommends swing-out centrifuges and higher speed (g) for better cell separation.
If not using a plasma separator tube with an activated gel barrier, pipette the plasma into a clean plastic screw-cap vial and attach the label. Do not transfer red cells to the vial. Screw cap on firmly to prevent leakage.

^{*If using RCF to measure centrifugation, RCF = (RPM)2 × 1.118 × 10-5 × r, where r is the radius of the rotor (in cm).}

Common Causes for Unacceptable Blood Specimens and Inaccurate Test Results

Hemolysis

Hemolysis occurs when the membrane surrounding red blood cells is disrupted and hemoglobin and other intracellular components escape into the serum or plasma. Hemolyzed serum or plasma varies in color from faint pink to bright red, rather than the normal straw color. Grossly or moderately hemolyzed specimens may be rejected and even slight hemolysis may alter certain test results.

Hyperbilirubinemia

Icteric serum or plasma varies in color from dark to bright yellow, rather than the normal straw color. Icterus may affect certain determinations. Upon receipt of such specimens, Quest Diagnostics may request a new sample to assure results of diagnostic value.

Turbidity (Lipemia)

Turbid, cloudy, or milky serum or plasma (lipemic serum or plasma) may be produced by the presence of fatty substances (lipids) in the blood. Bacterial contamination may also cause cloudy serum. Moderately or grossly lipemic specimens may alter certain test results. A recent meal may produce transient lipemia; therefore, we recommend that patients fast 12-16 hours before a blood specimen is obtained.

Radioisotope Interference

Diagnostic procedures or therapy involving radioactive compounds may invalidate radioisotope assays. Obtain specimens for anticipated radioisotope assays before administering isotopes to the patient.

Frozen Specimens

Serum or plasma specimens need to be frozen only if specifically stated in the specimen requirements. In these cases, it is essential to freeze the specimen as soon as it is separated from the cells. Always freeze specimens in plastic tubes unless specifically instructed otherwise.

Lay the tube at a 45° angle to avoid tube breakage caused by expansion during freezing. Do not freeze plastic Serum Separator Tubes. An exception is a specimen submitted in a PPT tube; the plasma can be frozen and transported in the original tube.

Extreme cold may cause ordinary plastic labels to become brittle and detach from the specimen tube. Use clear tape to secure label to specimen transport tube.

Note: If more than one (1) test is requested on a frozen specimen, split the sample prior to freezing. Use a separate test requisition when submitting a frozen specimen; frozen and non-frozen specimens must not be submitted on the same test requisition. Indicate on specimen container and test requisition if specimen is plasma or serum.

If more than one (1) test is ordered on a single frozen sample and tests cannot be performed at the same time or in the same location, Quest Diagnostics will call the client to choose the test to be performed before testing can proceed.

Random Urine Collection

The normal composition of urine varies considerably during a 24-hour period. Most reference values are based on analysis of the first morning voided urine. This specimen is preferred because it has a more uniform volume and concentration, and its lower pH helps preserve the formed elements.

To reduce contamination, the specimen submitted for urinalysis should be a clean catch “mid-stream” sample.

Submit a first morning voided specimen whenever possible. Urine for pregnancy testing should be first morning void, or a random specimen with a specific gravity of at least 1.010. Note the time of collection of the specimen on the test requisition and on the label of the container. For urine chemistry tests, the 24-hour urine collection is the usual standard. For some of these tests, there are dietary restrictions that must be observed. For others, there are drugs that must be avoided prior to obtaining the specimen. This information is included as part of the specimen requirements for the individual tests in the Test Directory.

Note: Specimens for Urinalysis must be submitted in a yellow/red swirl-top preservative tube or yellow-capped UA pour-off tube. See Urinalysis test for specific information.

If a frozen specimen is required, freeze the urine immediately after collection. Pack in dry ice for transport to the laboratory.

24-Hour Urine Collection

Because proper collection and preservation of 24-hour urine specimens are essential for accurate test results, patients should be carefully instructed in the correct procedure.

Important Note: For those analyses requiring the addition of 6N HCl, add the acid at the start of collection. Have the patient collect each voiding in a smaller container and carefully pour the urine into the 24-hour container to avoid any possible acid burns to the patient (make sure the patient understands the hazard presented by the acid preservative). Be sure to mix urine thoroughly before removing the aliquot.

Follow these instructions if someone other than the patient is to collect the urine:

Unless the physician indicates otherwise, instruct the patient to maintain the usual amount of liquid intake, but to avoid alcoholic beverages.
During the collection period, place the 24-hour urine container (with appropriate preservatives, if applicable) provided by Quest Diagnostics in a refrigerator or cool place to prevent growth of microorganisms and possible decomposition of urine constituents. (See specimen requirements for the individual tests in the General Test Listing section for any information on required preservatives.)
Have the patient empty his/her bladder in the morning into the toilet (not to be included in the 24-hour collection). Write the date and time of voiding on the container label.
Collect the patient’s next voiding and add it as soon as possible to the 24-hour container.
Add all subsequent voidings to the container as in (4). The last sample collected should be the first specimen voided the following morning at the same time as the previous morning’s first voiding.
Mix the contents of the container gently but thoroughly. Examine to ensure that the contents appear homogeneous.
Measure and note the total volume of urine.
Transfer the required aliquot to the plastic screw-cap plastic containers provided by Quest Diagnostics.
Record the total 24-hour urine volume on the specimen container and on the Test Requisition before sending to the laboratory.
If required, refrigerate the aliquot until it can be sent to the laboratory. For frozen specimens, freeze before packing in dry ice for transport. (See section on FROZEN SPECIMENS.)
Ensure the lid is properly tightened to prevent leakage.

Follow these instructions if the patient is to collect the urine:

Important Note: For those analyses requiring the addition of 6N HCl, add the acid to the 24-hour container at the start of collection. Have the patient collect each voiding in a smaller container and carefully pour the urine into the 24-hour container to avoid any possible acid burns to the patient (make sure the patient understands the hazard presented by the acid preservative). Be sure to mix urine thoroughly before removing the aliquot.

Give the patient the clean, labeled container provided by Quest Diagnostics, and instruct the patient not to remove any sponge or preservatives (powder, liquid, or tablet) that may be in the container. Alert the patient that preservatives are hazardous chemicals and are not to be ingested.

Unless the physician indicates otherwise, instruct the patient to maintain the usual amount of liquid intake, but to avoid alcoholic beverages.
Instruct the patient to carry out steps 3-5 above and return the 24-hour collection to office for specimen pick-up.

Urine Preservative Chart

Timed Stool Collection

Carefully read the specimen requirements.
Collect timed specimens in a pre-weighed and sealable container (available from the laboratory upon request).
Determine weight of total sample.
Mix contents of timed sample well to obtain a homogeneous mixture.
Remove the required aliquot to a screw-cap plastic container and seal well.
Record the total weight and collection time of the sample on both the sample container and the test requisition. Do not send the entire collection unless instructions for specific test indicate otherwise.
For instructions for stool cultures, refer to the Test Directory.

Coagulation Sample Collection

3.2% citrate plasma, shipped frozen, is the only acceptable sample type. All other anticoagulants (heparin, EDTA, oxalate) are NOT acceptable.
Proper blood-to-anticoagulant ratio is required.
- Vacuette® tubes must be filled to completion to ensure the proper 9:1 blood to anticoagulant ratio is achieved.
- Routine collection requires 4.5-mL blood added to 0.5-mL sodium citrate. For patients with normal hematocrits, no adjustment is necessary. If the patient has a known hematocrit above 55%, adjust the amount of anticoagulant in the collection tube before drawing the blood according to CLSI Guidelines, below.
To avoid contaminating the sample with tissue thromboplastin or heparin, follow the guidelines below. These substances may alter results.
- Venipuncture must be clean, with no trauma.
- Hemolyzed samples are not acceptable.
- The first 5 mL of blood drawn from a patient should not be used for coagulation testing.
- If drawn through an indwelling catheter, flush with 5 mL of saline and discard the first 5 mL of blood collected before collecting the specimen for coagulation testing. Blood should not be collected from heparinized lines.
Mix the sample gently by inverting the tube gently at least but not more than four (4) times immediately after filling.
Process the sample as soon as possible (within 60 minutes). Do not store whole blood samples refrigerated prior to processing. Spin down the specimen at a speed and for an amount of time required to produce platelet poor plasma (<5,000 - 10,000/uL). This can be accomplished by centrifuging at 1500 x g for 15 minutes.
Preparing samples for transport:
- Transfer plasma into a plastic tube using a plastic Pasteur pipette. Do not use glass tubes or glass Pasteur pipettes as glass can activate the clotting cascade.
- Label each tube “plasma.” Submit a plasma aliquot for each coagulation assay requested (1 tube for each test). If possible, submit one (1) additional plasma aliquot for repeat and/or test additions.
- If requesting other tests that require serum on the same order, label these tubes as “serum.”
Ship samples for testing on dry ice. Samples must remain frozen in transit.
We highly recommend quick-freezing the sample to keep coagulation factors intact. This can be achieved by one of the following methods:
- Freeze with liquid nitrogen.
- Freeze in a mixture of dry ice and methanol.
- Freeze in a -70 °C freezer.