Pre-analytical Variables of Coagulation Testing


Vol. 12 •Issue 8 • Page 44
Pre-analytical Variables of Coagulation Testing

Standardization of the analytical process of lab testing has been widely accepted as a prerequisite for accurate, reliable and precise results. The pre-analytical phase of testing is equally important, but often falls outside the lab’s direct control.

The pre-analytical phase of coagulation testing has the potential to greatly impact test results. Factors such as phlebotomy, specimen handling and processing can directly affect the final result and, therefore, patient diagnosis and management.

The two most commonly performed assays in the clinical coagulation laboratory are the Prothrombin Time (PT) and the Activated Partial Thromboplastin Time (aPTT). Since these assays comprise the bulk of testing and form the basis of other tests such as factor assays and specialty testing (e.g., protein C and protein S), a brief introduction is warranted before delving into the specifics of pre-analytical variables.

The PT test as it is known today was described by Quick in 1935.1 It is an important screening test for the evaluation of bleeding disorders, but is more commonly used to monitor oral anticoagulant therapy (Warfarin). It is sensitive to quantitative or qualitative deficiencies of the coagulation proteins, factors II, V, VII, X and fibrinogen (Fig. 1). Deficiencies of any of these proteins will prolong the PT test, the extent of which is dependent on the level of deficiency and the reagent and instrument combination used to perform the test.

In 1953, Proctor and Rapaport described the aPTT;1 the test has changed little since then. The aPTT detects qualitative or quantitative deficiencies in the intrinsic pathway of coagulation (factors XII, XI, VIII, IX, prekallikrein and high molecular weight kininogen) and the factors participating in the common pathway (factors II, V, X and fibrinogen). The test is commonly used to monitor unfractionated heparin therapy, monitor replacement therapy and also is affected by certain inhibitors of coagulation, such as the Lupus Anticoagulant (LA). LAs are antibodies (IgG, IgM) that interfere with in vitro phospholipid-dependent clotting tests, such as the aPTT. Clinically, the identification of these antibodies is important since they are associated with recurrent fetal loss and arterial and venous thrombosis.

As with PT reagents, aPTT reagents also vary in their performance characteristics, specifically their sensitivity to factor deficiencies, heparin responsiveness and inhibitor sensitivity.

Pre-analytical variables that affect the PT, aPTT and the assays based on these tests can be broadly divided into two categories–specimen collection and specimen handling. Specimen handling also includes processing and storage.

Specimen Collection

Verification of patient identification is the first step in the collection of any patient sample. Knowledge or access to the patient history may be necessary, as many medications such as anticoagulant therapies (warfarin, heparin and direct thrombin inhibitors), blood product and component transfusion and coagulation factor replacement therapies all impact coagulation test results.

Over-the-counter drugs (e.g., aspirin) have profound effects on platelet function studies. In addition, the patient’s physiologic state plays a role. Recent thrombotic episodes are likely to decrease coagulation protein levels as a result of the clotting process, which may lead to an incorrect test interpretation as a protein deficiency. Conversely, certain clotting proteins may be increased (fibrinogen, factor VIII and von Willebrand factor) in acute illness or pregnancy and may mask deficiency.

The phlebotomy itself should be a standardized, non-traumatic procedure with patients drawn from a supine or sitting position. If possible, the tourniquet application should be less than one minute to avoid stasis. Since some coagulation proteins display circadian variation, the time of collection of tests for these proteins should be consistent.

With rare exceptions (such as serum fibrinogen degradation products), coagulation testing is performed using citrated plasma. The anticoagulant for testing recommended by the NCCLS is 3.2 percent trisodium citrate, buffered or nonbuffered.2 Sodium citrate rapidly binds calcium, making it unavailable to participate in clot formation.

Prior to the 1998 NCCLS guidelines on collection, transport and processing of blood specimens, two citrate concentrations were acceptable for coagulation testing–3.2 percent and 3.8 percent. With a higher citrate concentration, more calcium ions are bound and longer clot times are obtained. The normal ranges are minimally affected; however, differences are more pronounced with samples from patients on anticoagulant therapy and when testing is performed with more sensitive reagents.3

The problem is further amplified when tubes are underfilled, upsetting the optimal blood to anticoagulant ratio, or if there is marked variance in the hematocrit.4 To eliminate this potential variation, the most recent NCCLS guidelines2 recommend the use of one citrate concentration, 3.2 percent, with the overall aim of improving standardization, specifically regarding PT testing and oral anticoagulant therapy monitoring.

Commercial tubes are available for coagulation testing and the majority of laboratories purchase these. Citrate tubes are available either in glass (with a non-activatable surface) or plastic. Environmental and safety concerns have led many labs to evaluate the plastic citrate tubes that have recently become available. If the decision is made to switch from one type of tube to another, it is important to ensure equivalent performance using both normal and abnormal samples.

Regardless of the type of tube used, the blood to anticoagulant ratio is 9:1 (nine parts blood to one part anticoagulant). Inadequate filling will increase the citrate-to-blood ratio and may lead to prolonged clotting times. The point at which underfilling begins to affect results is dependent on the specific reagent in use.

Another factor that affects the final citrate concentration is the hematocrit. With a high hematocrit (>55 percent), less anticoagulant is required. The calculation used to adjust the citrate concentration is shown in Fig. 2. There is no recommendation for adjusting the citrate concentration in patients with a hematocrit less than 20 percent.

A blood collection system that collects blood directly into a tube containing the anticoagulant (i.e., VacutainerTM system) is preferred over a syringe draw (recommended needle gauge 19-22 for adults and 21-23 for pediatrics). Aside from the potential safety concerns associated with a syringe draw, there is an increased risk of clotting within the syringe and hemolysis. Hemolyzed samples are not acceptable for coagulation testing, as clot-promoting substances may be released from the broken cell membranes. In addition, the hemolysis may interfere with analyzers utilizing a photo-optical method for clot detection. Unfortunately, there is no data quantifying the level of hemolysis that is acceptable; the decision must be made based on experience and the situation at hand.

If a syringe draw is unavoidable, a small volume (<20 mL) syringe is recommended so that clotting in the syringe during phlebotomy is avoided. If samples must be obtained from a catheter, heparin contamination and dilution must be considered. 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.

Further, the specimen should not be collected from heparinized lines. If questionable results are obtained from samples collected through a catheter, a new sample drawn from a different site should be requested before an expensive and lengthy laboratory investigation is begun. If a catheter sample is the only option and heparin contamination is suspected, the sample should be treated with a resin that neutralizes the effects of heparin. A sample collected from a catheter is unacceptable if monitoring heparin therapy is required.

According to the NCCLS guidelines on specimen collection, if multiple blood samples are collected, the coagulation tube should be the second or third evacuation tube, preferably after a non-additive tube.2 This is to avoid potential tissue thromboplastin or additive contamination. A non-additive discard tube should be drawn before the coagulation tube when only a coagulation specimen is required. However, the use of a discard tube increases the cost to the institution and obtaining the additional blood volume may be difficult in certain patient populations.

The necessity of the discard tube was addressed by McGlasson and coworkers.5 They compared PT and aPTT results obtained between the first and second evacuation tube using three different reagent and instrument combinations. No clinical or statistically significant differences were obtained between the first and second tube, suggesting that the NCCLS guideline for the use of a discard tube may need to be reconsidered.

Specimen Transport, Processing, Storage

Once the specimen is obtained and gently mixed by inversion, it should be transported to the laboratory as soon as possible for testing. It formerly was recommended that samples should be placed on ice following phlebotomy. However, this practice is controversial, as there is concern regarding cold activation of factor VII and the potential for shortening of clotting times. The current NCCLS guidelines do not recommend this practice.2

Plasma is obtained by centrifugation at a speed and time that produces platelet-poor plasma (<10,000 platelets/mL), approximately 15 minutes at 1500 g at room temperature. A centrifuge with a swing out bucket is preferred; this minimizes the remixing of plasma and red blood cells.

Upon platelet activation, platelets release a protein called platelet factor 4 (PF4), a potent heparin binding and neutralizing protein. The presence of platelets in the plasma sample has the potential to interfere with heparin therapy monitoring (PF4 neutralization of heparin will result in falsely low aPTT and heparin assay results). In addition, platelet contamination interferes with lupus anticoagulant testing, as the phospholipid provided by the platelet membrane neutralizes the effects of these antibodies. The effect is further exacerbated if the sample undergoes a freeze-thaw cycle, which ruptures the platelets, or if the plasma is allowed to sit on the cells for an extended period of time.

The time interval between collection of the sample and testing is dependent on the temperature of storage, the testing required and whether the patient is on unfractionated heparin (Table). The shortest time interval is required for aPTT testing on samples from patients on unfractionated heparin therapy. The rationale for this is to minimize heparin neutralization by platelet PF4 as discussed. These samples must be centrifuged within one hour and tested within four hours from time of specimen collection. If agitation of the specimen is likely to be encountered, such as during extended transport or from lines of automation, the plasma should be removed from the cells within one hour of collection and tested within four hours.

If testing cannot be completed within the stated time intervals, plasma should be frozen at Ð20¡ C for up to two weeks or at Ð70¡ C for up to six months. Frost-free freezers should be avoided, as the cycling of temperature that is used to eliminate frost build-up will result in repeated thawing and freezing of the samples. Thawing of the samples should be done quickly (< five minutes) at 37¡ C with gentle mixing and testing performed immediately, or within two hours if the samples are kept at 4º C.

Several investigators have assessed the stability of coagulation proteins in frozen plasma that would support the extension of these guidelines. In one study, Woodhams and coworkers6 evaluated the stability of all the commonly measured coagulation factors on samples stored at -24º C and -74º C. They reported that the aPTT, PT, thrombin time, fibrinogen and clotting assays for the individual factors were stable for up to three months if frozen at -24º C or lower. Freezing at -74º C extended the stability up to 18 months. They also observed that the best stability was obtained when small samples (1 mL) were stored in screw-cap tubes with a minimum of dead space.

Conclusion

It is important to be aware of the pre-analytical factors that may impact test results, not only to provide accurate patient data, but to avoid costly and time-consuming laboratory workups or delays in medical procedures from incorrectly collected specimens.

Dr. Worfolk is U.S. Scientific Manager, Diagnostica Stago, Parsippany, NJ.

References

1. Owen C. Tests of Hemostasis. In: A History of Blood Coagulation. Rochester, MN: Nichols W, Bowie E., editors. 2001, p.189-220.

2. NCCLS. Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays; Approved Guideline – Third Edition. 1998, H21-A3, Vol. 18, No. 20.

3. Adcock D, Kressin D, Marlar R. Effect of 3.2% vs. 3.8% sodium citrate concentration on routine coagulation testing. AJCP 1997;107:105-110.

4. Adcock D, Kressin D, Marlar R. Minimum specimen volume requirements for routine coagulation testing: Dependence on citrate concentration. AJCP 1998;107:595-599.

5. McGlasson D, More L, et al. Drawing specimens for coagulation testing: Is a second tube necessary? Clinical Laboratory Science 1999;12:137-139.

6. Woodhams B, Giradot O, et al. Stability of coagulation proteins in frozen plasma. Blood Coagulation & Fibrinolysis 2001;12:229-236.

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