Specimen Collection

Coagulation Testing

Coagulation testing is highly sensitive to a myriad of specimen collection and handling variables.  Because important diagnostic and therapeutic decisions are based on the results of coagulation tests, a procedural guideline is required to fully address these variables.  The following information applies to all Routine Coagulation tests (i.e., Prothrombin Time, APTT, Fibrinogen, etc.).  For details on Special Coagulation tests (Lupus Panel, DRVVT, Platelet Aggregation, etc.) refer to the individual test procedure or the Laboratory Survival Guide.

COLLECTION TUBES

  • It is highly recommended that blood specimens for coagulation testing be collected by venipuncture using a vacuum collection device that collects the specimen directly into an evacuated tube with a non-wettable surface.

  • For those samples collected from a normal saline lock (capped off venous port), twice the dead space volume of the catheter and extension set should be discarded.

  • 3.2% tri-sodium citrate (light blue-top) is the proper anticoagulant. This is the anticoagulant recommended by Clinical and Laboratory Standards Institute (formerly NCCLS) H21-A3 Guidelines. This laboratory requires the use of 3.2% tri-sodium citrate for all coagulation testing. If any 3.8% citrate tubes are received, the test is cancelled and the physician is notified.  No other anticoagulants are acceptable for coagulation testing.

  • Light blue-top tubes (citrate) are available in a 4.5ml full draw tube or a 2.7ml and 1.8 ml draw to accommodate pediatric testing volumes. Partial draw tubes are NO longer acceptable due to manufacturer's recall.

  • These tubes are pre-calibrated to draw the specified amount of blood, resulting in the proper 9:1 ratio of blood to anticoagulant.

 Examples:

Hemoguard Closure tubes, 4.5mL This is the "preferred" tube for most Coagulation testing.  It is a plastic tube that is identical to the conventional glass or plastic 4.5 mL vacutainer tubes, with the added safety feature of a protective plastic cover over the rubber stopper.  These tubes can be placed directly on Coagulation instrument after centrifugation.

Conventional Closure tubes, 4.5 mL:  Conventional closure glass vacutainer tubes are still in circulation, but are not the recommended tube for Coagulation testing. 

Conventional Closure Pediatric tubes, 2.7mL, 1.8 mL Conventional closure glass vacutainer tubes are available in pediatric volume sizes. 

 

NEEDLE SIZE

  • 20-21 gauge needles are recommended to avoid clotting or hemolysis.

  • For the pediatric patient, a 21- to 23-gauge needle may be used.  A winged blood collection set of the same gauge can also be used.

  • Syringe draws are discouraged because of the increased risk of hemolysis.  Additionally, with larger syringes, there is an increased chance that clotting may occur. 

  • If a syringe is used, a small volume syringe £ 20mL is recommended.

  • If a hypodermic needle/syringe must be used, it is vital that the blood be transferred to the vacutainer tube within one minute of completion of draw.

INDWELLING CATHETER COLLECTION

  • Under certain circumstances, blood specimens for coagulation testing may be drawn from an indwelling catheter.  In this case, the line should be flushed with 5 mL of saline and the first 5 mL of blood or six dead space volumes of the catheter discarded. 

  • Collection of blood through lines that have been previously flushed with heparin should be avoided, whenever possible.  If the blood must be drawn through an indwelling catheter, possible heparin contamination and specimen dilution should be considered.

  • If heparin contamination is suspected, the laboratory should be notified.  (Heparin Neutralization is possible in some situations.)  This method is discouraged due to a higher likelihood of obtaining a hemolyzed specimen which could result in specimen rejection.

TECHNIQUE

  • Specimens should be obtained from a single venipuncture with minimal tissue trauma. The blood should flow freely into the container.

  • Traumatic venipuncture and/or slow-flowing draws should be avoided.  Either may result in an activated or clotted sample.  Prolonged venostasis may raise the levels of factors VIII:C and IX, or it may activate the fibrinolytic system.

  • Regardless of the device used for specimen collection, all tubes should be gently inverted IMMEDIATELY  at least  five times to mix. DO NOT shake or mix vigorously.

  • If only a coagulation specimen is drawn, a pilot tube should be collected and discarded.   If multiple specimens are collected, the coagulation specimen should be collected into the second or third tube. Refer to CLSI recommendations (see below).

  • If a double syringe technique is used, blood from the second syringe should be used for the coagulation specimen. The number of specimen transfers and intermediate collection devices should be kept to a minimum.  Refer to CLSI recommendations (see below).

CLSI RECOMMENDED ORDER OF DRAW:

By following the following "order of draw," many pre-analytical errors and cross-contamination of additives will be avoided. 

TUBE TOP COLOR

1  YELLOW

(Culture tubes/vials)

2  RED

 (Non-additive tubes/serum tubes)

3  LIGHT BLUE

(Citrate tubes- Coagulation tests)

4  SPECKLED

 (Gel separator tubes)

5  GREEN

(Heparin tubes)

6  LAVENDER

 (EDTA tubes)

7  OTHER

(Other additives)

COLLECTION PROBLEMS

UNDERFILLING:  Inadequate filling of the collection tube will decrease the required blood:anticoagulant ratio (9:1), and may lead to falsely prolonged results.

OVERFILLING Overfilling will cause an incorrect blood to anticoagulant ratio which may result in slightly decreased results or the specimen may clot or contain fibrin due to inadequate anticoagulant for the blood volume drawn which will result in the specimen being rejected.

HIGH HEMATOCRIT Results on patients with a high hematocrit (³55%) may be falsely prolonged.  Refer to the section (below) on special handling of specimens with HIGH HEMATOCRITS.

CLOTTED SPECIMENS Clotting will lead to erroneous results.  CLSI recommends 5-10 inversions to prevent clotting.  Each tube is visually examined prior to centrifugation for the presence of obvious clots.  If in doubt, the specimen is checked for clots using applicator sticks.

HEMOLYSIS:  Hemolysis of the RBC's causes release of hemoglobin into the plasma, which shortens the APTT test.   Hemolysis also suggests the possibility of in vitro clotting, which would adversely affect ALL coagulation tests.  Therefore, samples that have visible hemolysis are rejected and a redraw requested. If the level of hemolysis is in question, the specimen will be processed using a "Downtime Label" to determine acceptability.

SPECIAL COAGULATION TESTS: CLSI guidelines recommends drawing a discard tube before specimens collected for special coagulation tests.

SPECIMEN TRANSPORT

CONDITIONS of TRANSPORT and STORAGE

Coagulation tests are enzymatic procedures and, as such, are subject to stringent time-frame and storage guidelines.  Reaction temperatures and the pH of specimens must be controlled at all times.  For best results, most sources recommend that specimens for Coagulation testing be delivered to the laboratory for testing within 1/2 hour of collection.  Considering the logistics and problems associated with transporting specimens, this recommendation is rarely achieved.  More realistic time-frame guidelines have been established and must be adhered to.  Receipt in lab beyond the stated guidelines will result in REJECTION of the specimen.  The allowable time interval between collection of the specimen and testing of the sample will depend on the transport temperature and the storage of the specimen.  Specimens for coagulation testing should be processed/stored as follows:

 Most specimens for routine Coagulation testing can be transported either as whole blood or centrifuged (plasma) form.  If plasma is sent, proper centrifugation protocol must be followed.

Specimens for routine Coagulation testing should be transported either at room temperature* (18-24°C) or refrigerated (2-4°C).  *Specimens for Prothrombin Time testing (PT) should be transported at room temperature.  They should NOT be refrigerated.

  • PT assays must be performed within 24 hours of collection.

  • APTT assays must be performed within 4 hours of collection.

  • ALL other COAGULATION tests must be performed within 4 hours of collection.

  • When samples cannot be assayed within the required time frame, the plasma must be separated from the red cells and frozen within one hour of collection.

Snap freezing platelet-free plasma (-70°C) is acceptable ONLY if performed by trained technical personnel.  There are inherent problems associated with proper freezing/ thawing techniques that can result in inaccurate test results.  If properly frozen (-70°C) and aliquotted (capped plastic cryo-tubes), plasma is viable for Coagulation testing for 6 months or at -20°C for 2 weeks. 

SPECIMEN PROCESSING

SPECIMEN IDENTIFICATION

All specimens received in this laboratory must be properly identified and be accompanied by an appropriate requisition or manifest.  Laboratory personnel are required to stringently check identification and test request information prior to processing specimens. Results will NOT be reported on any patient specimen if there is any doubt as to the validity of the requisition and/or the identity of the specimen.  

SPECIMEN QUANTITY vs TUBE SIZE

  • Sodium citrate tubes are pre-calibrated to draw a specific amount of blood to produce the proper 9:1 ratio of blood to anticoagulant. 

  • This ratio is critical in all methods of Coagulation tests.

  • There are various sizes of tubes available for use in Coagulation.  See COLLECTION TUBES (pg 1) of this document.

  • Each tube is checked for proper specimen amount prior to placement of LIS label and prior to centrifugation.  Volume 'comparison' tubes are available for all of the different tube types and sizes near the centrifuges. 

  • Specimens that do not have the proper amount of blood will be rejected. 

CHECKING FOR CLOTS

  • Each specimen is checked for the presence of clots prior to placement of the LIS label and prior to centrifugation. 

  • The tube is inspected for the presence of clots by careful observation while gently inverting the specimen. 

  • If a clot is suspected, the tube is uncapped, and checked with a pair of applicator sticks. 

  • Clotted specimens are rejected. 

LIS LABEL

After identification and test request is confirmed, volume is checked, and the specimen is checked for presence of clots, the tube is labeled with a Laboratory Information System (LIS) label.  LIS label information is checked against requisition slip and patient identification information prior to testing.

CENTRIFUGATION

For electromagnetic viscosity detection instruments (i.e., STAR, Compact), fast-spun platelet-poor plasma is preferred ( < 10,000/ul).  Routine Coagulation testing in this laboratory utilizes the STAT60 centrifuge to obtain this plasma.  Quality Control checks on platelet counts are performed on a semi-annual basis, and documented in the Centrifuge Maintenance log. 

  • Designed for rapid separation of blood cells from plasma or serum.

  • Speed:  7200 ± 350 RPM (4227 RCF)

  • STD mode:  3 minutes, Platelet-Poor Plasma

Note:  always invert specimen 2-3 times before centrifugation to facilitate achieving low residual platelet counts.

SPECIAL COAGULATION TESTS

 Special Coagulation tests or frozen plasmas require double spun Platelet-Free plasmas.

Laboratory Processing:

  • Spin in the Beckman centrifuge @ 4000 RPM for 10 minutes

  • Transfer plasma to 12x75 plastic tube

  • Cap tube and re-spin at 4000 RPM for 10 minutes

  • CAREFULLY remove from centrifuge without disturbing any platelets and/or cell pellets that might be on the bottom or sides of the tube.

  • Carefully transfer plasma into freezer tubes (plastic screw-top cryo-tubes) for testing or freezing (-70°C).

Special tests that require double spun to obtain platelet-free plasma are:

In-house Tests

  • Heparin Neutralization Procedure

  • Lupus Panel (Dilute Russell's Viper Venom Test)

  • AT III (Anti-Thrombin III)

  • APCR (Activated Protein C Resistance)

  • Bethesda and Factor Assays (if saved for later testing)

  • Heparin Anti-Xa Assay

Sendouts

  • PROTEIN C

  • PROTEIN S

Specimen Testing

After centrifugation, specimens are loaded onto the appropriate Coagulation Instrument (Stago:  STAR or COMPACT)  for Routine Coagulation  testing. 

  • All tubes are directly loaded (uncapped) into the cassettes.

  • Specimens received in 2.7 ml pediatric tubes should be uncapped and run on the COMPACT, or must have the plasma transferred to a microtainer.

  • 1.8 ml pediatric tubes must have plasma transferred to a microtainer prior to testing.

After all tests are run, the original collection tubes (and/or remaining transferred plasma) are refrigerated and retained (capped), for 24 hours.  Tubes are stored according to shift of testing.

If Special Coagulation tests are ordered in addition to the Routine test, laboratory personnel will refer to special handling instructions included in the individual test procedure.

After 24 hours, specimens are disposed of in specially designated RED BIOHAZARD BAGS/BOXES. 

High Hematocrits

It is the responsibility of the laboratory to detect any specimens received for coagulation testing that might have an elevated hematocrit. Specimens with extremely elevated hematocrits (³55%) require special handling for Coagulation testing due to improper plasma to anticoagulant ratio.
 

Polycythemia (high hematocrits) can cause falsely prolonged results.

When a specimen with a hematocrit  ³55% is identified, the laboratory will notify the attending RN or MD and prepare special tubes to be used for coagulation testing. 

 The lab has two mechanisms to detect specimens with high hematocrits:

  1. Comparison tubes are located next to the centrifuges (all 3 standard sodium citrate tube sizes).  Testing personnel are instructed to visually compare patient specimens to prepared tubes AFTER spinning and prior to testing.  Placement of barcode labels should be such that this comparison can be performed with ease.

  2. Automation (CBC) flagging criteria has been set to alert testing personnel of any specimens with high hematocrits (³55%).  Automation techs notify Coagulation techs of patients with high hematocrits.  The Laboratory Information System is checked for pending Coagulation testing.  This information is communicated verbally and via the Hematopathology Shift Report.

If a patient specimen is determined to have a hematocrit ³ 55%, a special tube is prepared by laboratory personnel to adjust the final citrate concentration of the specimen.  The method for tube preparation is cited below.  The goal is to achieve a blood : anticoagulant ratio of 9:1. 

Lab personnel will provide the tube(s) for collection.

FORMULA:  y = amount of whole blood required per 0.25 ml Sodium Citrate

                   y = [(             60              )  X  4.5 ] ¸  2

                             100 - Hematocrit

Preparation of "special" tubes for polycythemic patients (HCT ≥ 55%): 

  1. Determine the actual patient hematocrit. 

NOTE:  This can be accomplished by checking the LIS for concurrent CBC results, running original blue-top (citrate tube) on CBC analyzer [multiply result by 1.1 to compensate for blood:anticoagulant difference], or requesting that a new CBC be drawn.  One of the first two options is preferable.

  1. Determine the proper amount of patient blood (y) required to be added to 0.25mL citrate to achieve the proper 9:1 ratio.  This is done by using the formula (above) or chart (below).

  1. Prepare a COMPARISON TUBE

  • Place 0.25 mL of water in a small plastic tube (12x75)

  • Add an additional amount of water that is equal to the amount of whole blood determined in step #2 above (y)

  • Cap and label as COMPARISON tube

  1. Prepare PATIENT TUBE

  • Place 0.25 mL citrate in another 12x75 plastic tube.

  • Align this tube next to the COMPARISON tube prepared above.

  • Mark a "FILL" line on the PATIENT tube that is even (level) with the total volume in the COMPARISON tube. 

  • Cap and label as PATIENT tube.

  1. Give attending RN/MD the PATIENT tube.

  • Instruct RN/MD to ADD patient's blood to the "FILL" line of the PATIENT tube.

  • Cap tube and invert at least five (5) times.

  • Return PATIENT SPECIMEN TUBE promptly to the Hematopathology Lab for testing.

HEMATOCRIT (%)

AMOUNT OF BLOOD REQUIRED in 0.25 mL Citrate (mL)

55

3.0

56

3.1

57

3.1

58

3.2

59

3.3

60

3.4

61

3.5

62

3.6

63

3.6

64

3.7

65

3.9

66

4.0

67

4.1

68

4.2

69

4.4

70

4.5