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Ensuring Specimen Integrity:

Proper Processing and Handling of Specimens for Coagulation Testing


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October 2008

Preanalytic error and variability affects all laboratory tests but can be especially problematic for assessment of coagulation disorders. Collection, transport, processing, and storage of specimens for coagulation testing pose unique challenges. Often, preanalytic problems that should lead laboratory staff to question the accuracy of an abnormal result may not be readily apparent. Therefore, it is of the utmost importance that handling of coagulation specimens prior to analysis follows standardized procedures to ensure integrity.

Specimen Collection

During the collection process, the very act of the venipuncture may produce spurious activation of the hemostatic system. A clean, nontraumatic venipuncture minimizes this effect by limiting the introduction of tissue thromboplastin.

The collection system of choice for coagulation testing is an evacuated collection tube containing 3.2% dihydrate trisodium citrate (light blue-top), using an adapter with a 19 to 21-gauge needle.1 When other collection systems are used, precautions must be taken as described in Table 1.

Collection system Cautions

Winged infusion set (butterfly)


Used for neonates, children, or adults with problematic venous access

  • Air in collection set tubing results in an incorrect blood to anticoagulant ratio and adversely affects test results
  • Discard tube must be used to eliminate the air volume in tubing if coagulation specimen is the first, or only, tube drawn; alternately, a double syringe system may be used to remove air from the tubing
Syringe and needle set
  • Syringe must have nonactivating surface (eg, polypropylene)
  • Small volume syringe (≤20 mL) is recommended; larger syringes may allow clotting during the collection process
  • Blood must be transferred to collection tube and properly mixed within 1 minute of completion of draw
  • Rapid filling of collection tube may induce hemolysis
Small-gauge needle (25 or smaller) with either evacuated tube device or syringe
  • Blood drawn rapidly through small-gauge needle may cause activation of platelets and hemolysis of cells
Syringe or evacuated tube system and vascular access device (VAD)
  • Air leakage in the system will cause hemolysis or underfilled collection tubes
  • Avoid blood collection from heparin-flushed lines to avoid heparin contamination and specimen dilution
  • If heparin line must be used, flush line with 5-mL saline and discard the first 5 mL of blood (or 6 line volumes) of blood

Table 1. Collection System Cautions1

Once initiated, blood flow should be uninterrupted, and the collection tube must be filled as full as the vacuum permits. In order to maintain a ratio of 9 parts blood to 1 part anticoagulant, the collection tube must be filled to at least 90% fill volume. Underfilling or overfilling results in an imbalance in the blood to anticoagulant ratio and can produce artificially prolonged or shortened clotting times, respectively.2–5

To maintain the 9:1 ratio, patients who have hematocrit values of <25% or >55% should have the anticoagulant volume adjusted appropriately to maintain the 9:1 ratio using the formula provided below:

Failure to adjust the anticoagulant volume in these specimens may lead to erroneous results.6,7

The specimen should be mixed immediately by gentle inversion (end over end 5–6 times) to fully integrate the anticoagulant with the blood. Vigorous shaking and continued inversion of the specimen must be avoided to limit platelet activation.

Specimen Transport

Proper specimen transport must be accomplished both within the hospital and as the specimen is transported within or between cities and states. Vibration, mode of transportation, position of tubes, time from collection to processing, and temperature are all capable of affecting specimen quality.

Time Delay and Short-Term Storage

Blood specimens for coagulation testing should be stored at room temperature in the unopened collection tube and, ideally, transported to the laboratory within 1 hour.1 When specimens are collected off-site, it is not always possible to accommodate this requirement. The acceptable time between collection and processing, and short-term storage temperature, varies depending on the coagulation test performed and whether the patient is receiving unfractionated heparin therapy. General recommendations are outlined below in Table 2.

Formula:
anticoagulant volume (mL) = (100 – hematocrit)/(595 – hematocrit) x volume of specimen


  Whole blood specimen (unopened) Processed and aliquoted plasma (capped)
Room temperature Refrigerated Room temperature Refrigerated
Prothrombin time (PT) Up to 24 hours Unacceptable Up to 24 hours Unacceptable
Activated partial thromboplastin
time (APTT) (nonheparin)
Up to 4 hours Unknown 4 hours 4 hours
Activated partial thromboplastin
time (heparinized)
1 hour Unknown 4 hours 4 hours
Special coagulation testing 4 hours Unacceptable 4 hours 4 hours

Table 2. Acceptable Time Delay and Short-term Storage for Coagulation Test1,8

Temperature

Whole blood specimens for coagulation testing should be transported at room temperature, avoiding high and low temperature extremes. Elevated temperatures accelerate degradation of coagulation factors V and VIII, while prolonged specimen exposure to cold can activate factor VII.

Mode of Transportation, Vibration, and Position of Tubes

Specimens within a hospital setting may be hand-carried or sent via pneumatic tube to the laboratory. When collected off-site, specimens may be transported by courier. In both situations, specimens should remain upright (vertical orientation) and care must be taken to protect the specimens from vibrations and movement to minimize protein denaturation and platelet activation.1 When transporting by car, the temperature inside the vehicle must be strictly controlled. Samples that are sent to Mayo Medical Laboratories via airplane must be packaged to ensure consistent temperature inside the transport container. Frozen plasma samples must be packaged in a Styrofoam container with sufficient dry ice to ensure that they arrive frozen (ie, at least 5 lbs of dry ice).

Specimen Processing

Each laboratory must establish a standardized procedure for coagulation specimen processing to assure accurate and reliable results. Examine both the whole blood specimen prior to centrifugation, and the plasma sample after centrifugation. Centrifuge the specimen according to recommended guidelines and, finally, store the plasma sample at proper conditions as outlined in Table 2 to maintain the integrity of the sample until testing is completed.

Visual inspection of the specimen

Before centrifuging, check the whole blood specimen for proper fill and gross clot formation by gently inverting the collection tube. The tube must be at least 90% filled. If clots are observed, the specimen must be discarded and recollected because results will be unreliable. However, small fibrin clots will not be detected at this time and may appear only after thawing a previously frozen sample. After centrifugation, examine the plasma for fibrin clots and pour the cellular portion through gauze to observe for small red cell clots. Samples with fibrin or red cell clots are not suitable for coagulation testing and must be rejected. Inspect the plasma sample for excess hemolysis, icterus, and lipemia. Any one of these conditions may affect the accuracy and reliability of the test results and should always be noted. Samples that are grossly lipemic or icteric may interfere with light transmittance on some coagulation instruments. Hemolysis may cause coagulation factor activation and impact clotting time results.9,10 The Clinical and Laboratory Standards Institute (CLSI) recommends that grossly hemolyzed samples should not be used for coagulation testing.1 Depending on the severity, samples with any of these characteristics may be rejected by our laboratory staff.

Centrifugation

Proper centrifugation is necessary for obtaining plasma for coagulation testing. Specimens must be centrifuged at a speed and time that consistently produces platelet-poor plasma (<10,000 plts/µL). It is generally recommended to centrifuge capped whole blood specimens for coagulation testing at 1500 x g for no less than 15 minutes at room temperature.1 The centrifuge should be a swing-out bucket rotor type to minimize contamination of the plasma with platelets and other cellular components, especially when removing plasma to be frozen. To ensure that the plasma is platelet poor, the sample must be centrifuged a second time. Following the initial centrifugation process, use a plastic pipette to transfer the plasma to a plastic tube. Do not pour the plasma into the plastic tube as this may lead to cellular contamination of the plasma sample. Centrifuge a second time for 10 minutes. Remove the top portion of plasma, leaving approximately 500 μL in the bottom to discard. The double-centrifuged plasma should be aliquoted (1.0 to 2.0 mL each) into properly labeled plastic tubes.

Storage and Handling of Plasma Sample

If the plasma sample will be used to perform routine coagulation testing on-site, refer to Table 2 for short-term storage recommendations. When preparing a plasma sample to be sent to a reference laboratory for special coagulation testing, the sample must be frozen. Plasma aliquots may be frozen at -20ºC or below for up to 2 weeks or, preferably, at -70ºC or below, and must remain frozen during storage and shipment. It is important that a frost-free freezer not be used. The freeze-thaw cycle, in which the temperature is cycled several times during the day, causes coagulation factor deterioration.

Major preanalytic variables and their adverse effects on coagulation testing results are shown in Table 3.

Preanalytic Difficulty
Immediate Effect
Coagulation Test Result Consequence
Small-bore needle
  • Hemolyzed sample
    • light transmittance interference
  • Coagulation factor activation
  • Platelet activation
  • Unreliable results
  • Shortened clotting time results
  • Shortened APTT results in heparinized patients
Traumatic venipuncture
  • Introduction of tissue thromboplastin
  • Clotted specimen
  • Shortened clotting time results
  • Unreliable results
Inadequately mixed specimen
Clotted specimen
Unreliable results
Excessively mixed specimen
  • Coagulation factor activation
  • Platelet activation
  • Fibrin strands in plasma sample after thawing
  • Shortened clotting time results
  • Shortened APTT results in heparinized patients
Failure to correct for high hematocrit
Prolonged clotting time results
Underfilled collection tube
Prolonged clotting time results
Overfilled collection tube
Clotted sample
  • Shortened clotting time results
  • Unreliable results
Double centrifugation not done when preparing a sample to be frozen
Lysis of residual platelets upon freezing of plasma sample
  • False-negative lupus anticoagulant results
  • Shortened APTT results in heparinized patients
Plasma stored in frost-free freezer
Degradation of coagulation proteins
Low levels of most measured coagulation analytes
Prolonged storage at room temperature
Degradation of labile coagulation factors V and VIII
Prolonged clotting time results
Refrigerated collection tubes
  • Cold activation of factor VII
  • Cryoprecipitate of factor VIII and VWF
  • Shortened PT
  • Low levels of factor VIII and VWF

Table 3. Effects of Preanalytic Difficulties

Summary

Coagulation testing is especially prone to inaccuracies and misinterpretations due to the poor specimen quality. In order to optimize the accuracy and reliability of coagulation testing, preanalytic processes must be standardized according to recommended guidelines and strictly followed. In doing so, the laboratory will assist in ensuring correct patient diagnosis, management and therapy. For testing performed by Mayo Medical Laboratories, specific collection, processing, transportation, and storage instructions are available online and in our Test Catalog. Also provided in this issue is an insert that can be used in your laboratory as a guide for specimen processing of coagulation specimens.

Authored by Plumhoff EA, Masoner D, Pruthi RK, Dale JD

References

1.Clinical and Laboratory Standards Institute. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline—Fifth Edition. CLSI document 2008;H21–A5:Vol 28 No 35

2. Chuang J, Sadler MA, Witt DM: Impact of Evacuated Collection Tube Fill Volume and Mixing on Routine Coagulation Testing Using 2.5-mL (Pediatric) Tubes. Chest 2004;126:1262–1266

3. Reneke J, Etzell J, Leslie S, et al: Prolonged Prothrombin Time and Activated Partial Thromboplastin Time Due to Underfilled Specimen Tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol 1998;109(6):754–757

4. Peterson P, Gottfried EL: The Effects of Inaccurate Blood Sample Volume on Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT). Thromb Haemost 1982;47(2):101–103

5.Adcock D, Kressin DC, Marlar R: Minimum Specimen Volume Requirements for Routine Coagulation Testing: Dependence on Citrate Concentration. Am J Clin Pathol 1998;109(5):595–599

6. Marlar RA, Potts RM, Marlar AA: Effect of Routine and Special Coagulation Testing Values of Citrate Anticoagulant Adjustment in Patients With High Hematocrit Values. Am J Clin Pathol 2006;126:400–405

7. Siegel JE, Swami VK, Glenn P, Peterson P: Effect (or lack of it) of severe anemia on PT and APTT Results. Am J Clin Pathol 1998;110(1):106–110

8.Adcock D, Kressin DC, Marlar R: The Effect of Time and Temperature Variables on Routine Coagulation Tests. Blood Coag Fibrinol 1998;9:463–470

9. Laga AC, Cheves TA, Sweeney JD: The Effect of Specimen Hemolysis on Coagulation Test Results. Am J Clin Pathol 2006;126:748–755

10. Lippi G, Montagnana M, Salavgno GL, Guidi GC: Interference of Blood Cell Lysis on Routine Coagulation Testing. Arch Pathol Lab Med 2006 Feb;130(2):181–184


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