Specimen Collection

Common Collection Errors

Avoiding Common Collection Errors

Careful attention to routine procedures can eliminate most of the errors outlined in this section. Materials provided by the laboratory for specimen collection can maintain the quality of the specimen only when they are used in strict accordance with the instructions provided. To ensure a sufficient quantity of each type of specimen indicated for the procedures to be performed, please consult the volume requirements published in the LSG.

General Collection Errors

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

  • Failure to label a specimen correctly and to provide all pertinent information required on the test request form. (See Blood Chemistry and Hematology - Blood Collection/Transport Containers.)
  • Insufficient quantity of specimen to run test or QNS (quantity not sufficient). (See 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.

Serum Preparation Errors

The most common serum preparation errors include:

  • Failure to separate serum from red cells within 60 minutes of venipuncture.
  • Failure to allow the specimens to clot before centrifugation. (See Preparing Serum on clotting and serum-separator tubes and red-stopper tubes.)
  • Hemolysis: red blood cells break down and components spill 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).

Plasma Preparation Errors

The most common errors in the preparation of plasma include:

  • Failure to collect specimen in correct additive.
  • Failure to mix specimen with additive immediately after collection.
  • Hemolysis or damage to red blood cells breakdown.
  • Incomplete filling of the tube, thereby creating a dilution factor excessive for total specimen volume (QNS).
  • Failure to separate plasma from cells within 30-45 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.

Urine Collection Errors

The most common urine collection errors include:

  • Failure to obtain a clean-catch, midstream specimen.
  • Failure to refrigerate specimen or store in a cool place.
  • Failure to provide a complete 24-hour collection/aliquot or other timed specimen.
  • Failure to add the proper preservative to the urine collection container prior to collection of the specimen.
  • Failure to provide a sterile 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.

Hemolysis

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.

Grossly hemolyzed samples will be rejected. A sample visibly hemolyzed will be rejected for the following analytes: Acid Phosphatase, Alkaline Phosphatase Isoenzymes, Alkaline Phosphatase, Amylase, Amylase Isoenzymes, ALT, AST, Bilirubin, CK Isoenzymes, CK-MB, CPK Folate, Glucose, Lipase, LDH, LDH Isoenzymes, Phosphorous, Potassium, RPR, Type and Crossmatch, Type and Screen, VDRL(CSF), Alpha-fetoprotein.

Hemolysis can be caused by:

  • mixing additive tubes too vigorously or using rough handling during transport

  • drawing blood from a vein that has a hematoma
  • pulling back the plunger on a syringe too quickly
  • using a needle with too small of a bore for the venipuncture
  • using too large a tube when using a small diameter butterfly needle
  • frothing of the blood caused by improper fit of the needle on a syringe.
  • forcing the blood from a syringe into an evacuated tube
  • Excessive fist clinching
  • Leaving the tourniquet on for longer than one minute

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

  • For routine collections, use a 20- 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.)
  • If there is air leakage around the needle or loss of vacuum in the tube, replace the vacuum tube.
  • If you are using your own collection equipment instead of the vacuum tube technique, use only clean, dry, sterile needles, syringes, and tubes.
  • Collect blood in room temperature containers unless the specimen requirement specifies otherwise.
  • 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.
  • 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.
  • Premature removal of the tube causes a rush of air to enter the tube, which may result in damage to the red cells.
  • 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.
  • Allow collection site to dry after cleaning. Alcohol used to clean the puncture site may cause contamination in a tube.
  • Do not collect a specimen in a hematoma.
  • Allow specimen to clot completely before centrifuging.
  • Do not centrifuge the specimen for a prolonged period of time.

Proper Collection of Tubes with Anticoagulant

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

Blood collection tubes with anticoagulant should be inverted gently as soon after collection as possible to prevent clotting. All blood collection tubes must be filled to the fill line in order to prevent dilution of blood components. Tubes with anticoagulant improperly filled will be rejected.

Deliver the samples to the laboratory promptly. Valid measurement of analytes in serum or plasma requires prompt separation from the blood cells and analysis in the laboratory. When left unseparated, analytes shift between the cells and the plasma or serum and glucose, is consumed. In addition, some analytes are unstable at room temperature. Microbiology specimens require specific preservation methods according to body site. Refer to specific test for details.

  • Tap the tube gently at a point just below the stopper to release any additive adhering to the tube or stopper.
  • 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.
  • To ensure adequate mixing of blood with the anticoagulant or preservative, use a slow rolling wrist motion to invert the tube gently five or six times. Failure to invert tubes may lead to the formation of microscopic clots. Rapid wrist motion or vigorous shaking may contribute to hemolysis.
  • 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.
  • 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 serum-separator tube is an additive tube and should be inverted five to six times after collection. Allow the tube to stand 15-30 minutes for complete clotting to occur prior to centrifugation.

Vacuum Tubes without Anticoagulants

When using vacuum tubes containing no additives:

  • Permit the tube to fill completely.
  • Let the specimen stand for a minimum of 30 minutes and not longer than 60 minutes prior to centrifugation. (CLSI 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 altered.
  • Centrifuge the specimen at the end of the waiting period in strict accordance with the manufacturer's instructions for speed and duration of centrifugation (usually 10-15 minutes).

Specimen Clotted

Inadequate mixing of the vacutainer tubes as soon as possible after the phlebotomy will result in the blood not mixing with the anti-coagulant. By gently inverting the vacutainer tube 4-10 times, the blood will mix and clotting will not occur.

Quantity Not Sufficient

One of the most common and expensive errors 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 additional expense and inconvenience to the patient and to the physician. To ensure an adequate specimen volume:

  • Always draw whole blood in an amount 2½ times the required volume of serum required for a particular test.

For example, if 4 mL of serum are 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 serum-separator tubes. If pediatric tubes are used, be sure to collect an adequate volume of specimen to perform the test.

  • Provide patients with adequate containers and instructions for 24-hour urine and stool collections.
  • For most serum and plasma tests, check to be certain that the transport tube is half full. Note: Certain tests (eg, prothrombin time) require a 90% to 100% full tube in order to achieve the proper blood-to-anticoagulant ratio; otherwise, the specimen may be found to be QNS.