SPECIMEN COLLECTION

Biopsy Tissue | Blood Cultures | Coagulation | CSF | Cytogenetics | Cytopathology | Fecal | Pap Smears | Throat Culture | Urine Collection | Viral | Wound Sample

Proper sample collection and handling is an integral part of obtaining a valid and timely laboratory test result. Specimens must be obtained using proper phlebotomy techniques, collected in the proper container, correctly labeled (in the presence of the patient) and promptly transported to the laboratory.  It is the policy of the laboratory to reject samples when there is failure to follow these guidelines.  All specimens should be handled with universal precautions, as if they are hazardous and infectious.

Prior to each collection, review the appropriate test description, including the specimen type to be collected, the volume, the procedure, the collection materials, and the storage and handling instructions.

Specimen Collection Timing

The basal state (the early morning approximately 12 hours after the last ingestion of food) is recommended for determining the concentration of body constituents such as glucose, cholesterol, triglycerides, electrolytes, and proteins.

Blood composition is significantly altered after consuming food, and consequently alters many clinical chemistry tests. If a patient has eaten, and the physician still wants the test, then "non-fasting" is written on the request so the laboratory can make a notation on the report as to why some of the test values may be different than expected.  For outpatients, provide the patient in advance with appropriate collection instructions and information on fasting, diet, and medication restrictions when necessary.

Specimens for Coagulation Testing

Specimens obtained for Coagulation testing must be collected and transported to the laboratory according to strict guidelines in order to assure accuracy of results.   Refer to Coagulation Collection and Handling Guidelines for further instructions concerning requirements for Coagulation specimens.

1. Knock before entering the patient's room.

2. Introduce yourself

3. Identify patient using two patient identifiers.

4. Explain the procedure you will be performing.

Correct patient identification before specimen collection is extremely important.  Identify the patient prior to sample collection, using at least two patient identifiers.

Inpatients: Patients in the hospital should be wearing an identification bracelet that includes their last and first name, date of birth and a unique hospital number.  Proper identification should include a three-way match using information on the ID bracelet and the test requisition, and the patient's stating of his or her name.  If the patient does not have an ID bracelet, ask the nurse responsible for the patient to positively identify the patient and to place and ID bracelet on the patient.  For unconscious or unidentified patients, it is important a unique number or identification system be used.

Outpatients: For an outpatient or ambulatory setting, there is no ID bracelet, but the patient should have been given identification labels when he/she registered.  This label can be used along with asking the patient his/her name.  If there is no label, then another means of identification should be used.

All specimens submitted to Pathology Clinical Services for testing must be appropriately labeled to assure positive identification and optimum integrity of patient specimens from the time of collection until testing is completed and the result reported.  In accordance with standards issued by the College of American Pathologists (CAP), American Association of Blood Banks, and The Joint Commission, all specimens must be labeled at the time of collection; in the presence of the patient, to maintain identity throughout the pre-analytical, analytical, and post-analytical processes.  Refer to PCS Policy 7.01.03 Specimen Labeling Requirement for additional requirements.

If there is a question as to the integrity and/or identification of a sample, the laboratory will reject the sample and request recollection.  If extenuating circumstances exist that prevent recollection of the sample, and the physician or nurse requests that the test be performed on a sample that cannot be positively identified, the laboratory will analyze the sample with the following conditions:

• The ordering nurse or physician must come to the laboratory to personally identify and relabel the request slip and patient sample.

• The Improper Identification Release form must be completed and be legible. The correction must be dated and signed. Delegating this to another person will not suffice for medico-legal purposes.

Prioritize Collection:

"STAT" means special turnaround time and must be collected immediately. It may involve a patient whose medical condition has suddenly become very critical and must be treated as a medical emergency.

Fasting: Requests for "fasting" specimens are performed before routine requests, so patients can eat meals on schedule.

Order of Draw

Blood collection tubes must be drawn in a specific order to avoid cross-contamination of additives between tubes.  The recommended order of draw for plastic vacutainer tubes is:

1. First - blood culture bottle or tube (yellow or yellow-black top)

2. Second - coagulation tube (light blue top). If just a routine coagulation assay is the only test ordered, then a single light blue top tube may be drawn. If there is a concern regarding contamination by tissue fluids or thromboplastins, then one may draw a non-additive tube (red top) first, and then the light blue top tube.

3. Third - non-additive tube (red top)

4. Last - additive tubes in this order:

• SST (red-gray or gold top): Contains a gel separator and a clot activator.

• Sodium heparin (dark green top)

• PST (light green top): Contains lithium heparin anticoagulant and a gel separator.

• EDTA (lavender top)

• ACDA or ACDB (pale yellow top): Contains acid citrate dextrose.

• Oxalate/fluoride (light gray top)

Note: Tubes with additives must be thoroughly mixed.  Erroneous test results may be obtained when the blood is not thoroughly mixed with the additive. Mix all tubes with anticoagulant by gentle inversion for 15 seconds

(This is meant to be a guide and is not all-inclusive, if you have any questions, call the lab.)

Reference:  CLSI H3-A4 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard – Fourth Edition

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.

All laboratory specimens shall be placed in leakproof containers (i.e., culturettes, vacuum tubes), then bagged in single, biohazard specimen bags. Place the requisition slip in the outside pocket of the biohazard specimen bag.

Tubed Specimens: Specimens may be sent through the tube system as follows:

1. Place and seal the specimen in a biohazard bag, with the request slip in outside pocket.
2. Place the biohazard bag into a Zip N’ Fold pouch; completely sealing the pouch.
3. Load the Zip N’ Fold pouch into a pneumatic tube and send to the lab.

To ensure the validity of test results and the safety of laboratory personnel, specimens that leak in transit will be discarded, and the sender notified to resend another sample.

Spill Cleanup:  If a pneumatic capsule is received that appears grossly wet or soiled, wear gloves before handling the capsule and removing the contents. Be aware that there may be broken glass or plastic inside! Remove sharp objects (broken glass or plastic) using forceps. Discard any wet or soiled padding as infectious waste. Clean the inside and outside surfaces of the pneumatic capsule with a hospital-grade disinfectant. Call the physical plant dispatcher and notify them of the contamination of the tube system.

Shipping: DOT and IATA requirements will be followed when shipping specimens for reference testing.  Refer to PCS policy 3.18.16 - Packaging and Shipping of Infectious Substances and Diagnostic Samples for guidelines.

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