Coagulation and Pre-Analytical Variables

Coagulation test results are important in the clinical decision making process because they evaluate the functioning of the hemostatic process. Results are used to determine if a patient is at a risk to bleed or clot. Coagulation results are also used to as to monitor anticoagulant therapy of heparin and warfarin.

Pre-analytical variables are well defined in coagulation testing and account for a large percentage of error. Many issues arise due to the lack of standardization procedures for sample collection, handling and storage.

As a result, this accounts for up to 93 percent of errors within the entire diagnostic process.1 Specimen collection problems are the main cause of pre-analytical variables. Errors include samples that are hemolyzed–which is the most frequent sample collection error (54 percent), followed by insufficient samples (21 percent), incorrect specimens (13 percent) and samples that are clotted (5 percent). 1

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Incorrect Specimen
Even with the modern technology of bar coding, patient identification and properly labeled tubes still are a source of error. Samples are still drawn into the wrong tubes, and mislabeled.

There are also patient variables that can influence coagulation results, such as age, sex, diet, smoking, alcohol, medications and stress. 2

Biological variation is the natural fluctuation of the body fluid constituents. This type of variation looks at two different components:

Inter-individual variability which is the variability due to the heterogeneity of physiologic influence among subjects and intra-individual variability which looks at variability in the same subject over time.3

For example, lower prothrombin time (PT) values were observed at night, whereas a peak PT value was recorded in the afternoon. Activated partial thromboplastin time results (APTT) showed a peak in the evening or at night, although the magnitude of variation was small (<10 percent).4

Insufficient and Clotted Samples
Inappropriate specimen quality and quantity account for more than 60 percent of preanalytical errors.1

Variables include the importance of proper filling of the blood tube and maintaining the 9:1 ratio of blood to anticoagulant. If the specimen is under-drawn there will be too much anticoagulant in the tube and coagulation results can be falsely prolonged.

This is also observed in patients with a high hematocrit which occurs in neonates, patients with polycythemia or patients that present with conditions that cause hemoconcentration.

Conversely, if a sample is overdrawn, there may be too much plasma and not enough anticoagulant, resulting in a clotted sample. Obtaining a good venipuncture eliminates the introduction of tissue factor which may activate the sample and falsely shorten results.5

Hemolysis
The College of American Pathologists Q-probe study determined that in-vitro hemolysis occuring during phlebotomy is the most frequent reason for specimen rejection.1

Hemolysis can occur in-vivo from a pathological condition such as autoimmune hemolytic anemia or a transfusion reaction; or in-vitro from improper specimen collection due to a wrong needle size, excessive mixing of the blood sample, inadequate storage temperatures or rough handling during specimen transport.6

Hemolysis can also occur when blood is drawn from a peripheral IV catheter.7

Hemolysis results in the leakage of intracellular analytes into plasma this produces with falsely elevated results, measurable concentration or dilutional effects which can invalidate laboratory results.

The presence of hemolysis can yield a wide variation in coagulation results. It can not be assumed that because of hemolysis results are either prolonged or shortened.

It has been speculated that hemolysis provides a source of tissue factor that can cause coagulation factors to activate and shorten results, or that the process of hemolysis competes with coagulation reagents and will prolong coagulation results.8

The Clinical and Laboratory Standards Institute (CLSI) guidelines for PT and APTT testing state that samples with visible hemolysis should not be used because of possible clotting factor activation and interference with end point measurement.9

Other Pre-Analytical Variables
Proper centrifugation is important to obtain platelet poor plasma. This is defined as plasma containing <10 x 10 9 /L platelets.

Platelets are a source of phospholipids and their presence in plasma can interfere with reagents which are phospholipid based by falsely shortening results.5

CLSI guidelines states that platelet counts >10 x 10 9/L are acceptable on fresh specimens for PT and APTT testing providing patients are not on heparin.

However, they are not suitable for other coagulation testing (e.g., lupus testing, heparin monitoring). Also, if plasma is to be frozen it is imperative the plasma be platelet free or the platelets will burst and falsely shorten results. Centrifuges should be checked every 6 months to ensure platelet counts are acceptable. Filtering samples to remove platelets is not recommended, since von Willebrand factor can also be removed.

It has also been noted that tourniquet placement can influence the concentration of several analytes. It is possible to have tourniquet induced variations in coagulation laboratory testing due to stasis time and the biochemical characteristics of the analyte.

Hemoconcentration contributes to increased concentrations of large molecules, cells and coagulation factors. It is important to minimize the impact of venous stasis.1

The age and storage of samples in an important issue in coagulation testing. Samples for PT testing are stable unspun, unopened at room temperature for 24 hours. Storing samples cold may activate Factor VII. APTT samples from unheparinized patients can remain at room temperature either centrifuged or uncentrifuged for as long as 4 hours.

Cold temperatures can cause a decrease in Factor VIIII. Coagulation testing for APTT assays on heparinized patients should occur within 1 hour. If testing can not occur within 4 hours, plasma from these patients should be removed from the red blood cells and frozen. If they are allowed to remain on the cells, platelet factor IV will bind to heparin and falsely shorten results.10

The importance of a very simple practice of mixing samples can be overlooked in the laboratory. Many proteins can settle and samples need to be mixed. In particular, if samples are frozen precipitation of proteins may occur and mixing is critical.

Accurate Results
Understanding how pre-analytical variables can impact coagulation results is important. Laboratories need to be aware of the impact of these variables when interpreting patient results. Taking steps to minimize these variables will aid in providing patients with accurate coagulation results.

References

  1. Lippi G, Guidi G, Mattiuzzi C, et al. Preanalytical variability: the dark side of the moon in laboratory testing . Clin Chem Lab Med 2006;44(4):358-365
  2. Lawrence J., The key role played by pre-analytical variables, Lab Notes, BD Vacutainer Systeme. 1999:1-4.
  3. Banfi G, Del Fabbro M. Biological variation in tests of hemostasis, Seminars in thrombosis and Hemostasis 2008;34(7):635-41.
  4. Banfi G, Del Fabbro M. Biological variation in tests of hemostasis, Seminars in thrombosis and Hemostasis, 2009;35(1):120-6.
  5. Lippi G, Franchini M, Montagnana M, et al. Quality and reliability of routine coagulation testing: can we trust that sample? Blood Coagulation and Fibrinolysis, 2006;17:513-519.
  6. Arzoumanina L. What is hemolysis? Tech Talk, 2003;2(2).
  7. Kennedy, Angermuller S, King R, et al. A comparison of hemolysis rates using intraveneous catheters versus venipuncture tubes for obtaining blood samples. Journal of Emergency Nursing, 1996;22:566-569.
  8. Laga AC, Cheves TA, Sweeney JD. Specimen hemolysis and coagulation testing : the effect of specimen hemolysis on coagulation test results, coagulation and transition medicine. American Journal of Clinical Pathology, 2006;126:748-755.
  9. CLSI Document H3-A4 Procedures for the Collection of Diagnostic Blood Specimens by venipuncture;Approved Standard, 4th ed. Wayne, PA: 1998.
  10. CLSI Document H21-A5, Collection, Transport and Processing of Blood Specimens for testing Plasma Based Coagulation Assays and Molecular Hemostasis Assays, Approved guideline. 5th edition. Wayne, PA: 2008.

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