Errors in Coagulation Testing

Vol. 23 • Issue 2 • Page 24

The Learning Scope

Coagulation testing is a basic staple in the laboratory. From rapid prothrombin time (PT) assays in the physician office to screening and monitoring of PT and activated partial-thromboplastin time (APTT) and D-dimer in a hospital laboratory to larger hemostasis and reference laboratories performing more in depth testing, millions of coagulation tests are done each day. Are we getting reliable, useful results?

Quality assurance requires that assays are examined to assess the pre-analytical, analytical and post-analytical factors that may or may not affect a test. Many assays also have biological considerations regarding time of draw and individual variability. While these factors are important considerations for all testing, they are particularly important for coagulation testing. Let’s examine why.

Pre-Analytical Variables

In coagulation testing, pre-analytic variables range from the simple to the complex. Each variable is important. First is patient identification. Each facility has standard measures in place for correctly identifying the person from whom blood is drawn. Typically, identification is accomplished with two identifiers, often the patient name and birthdate or medical record number. The most important point in this variable is to have the patient speak their name and birthday while the phlebotomist is checking the armband. If the patient is unable to respond, someone needs to verify the identity of the patient. Often family members with the patient can assist in this measure.

The order of the draw is a common topic and has changed over the years. Sodium citrate tubes should be drawn first and no waste tube is necessary. However, if the collector is using a butterfly needle, a waste tube should be collected. If the blood is collected in a syringe and transferred to a sodium citrate tube, the blood should never be forced or “squirted” into the tube. This will cause platelet and coagulation activation. Many hospitals and offices prefer coagulation studies not be drawn in a syringe due to the increased risk for hemolyzing the specimen.

The preferred anticoagulant is always sodium citrate and the recommended concentration is 3.2% with a whole blood to anticoagulant ratio of 9:1. This ratio prevents partially filled tubes from being accepted for testing. The volume of the tube should be at least 90% or results can be compromised due to excess anticoagulant.

Whenever possible, blood should be from a stick that was non-traumatic. A ðtraumatic stick, meaning the phlebotomist had to “probe around” searching for the vein, will compromise results, as will leaving the tourniquet on longer than one minute. Needle size is important and needles less than 25 gauge should be used. Specimens that are hemolyzed, icteric or turbid will produce invalid results in an optical measurement system. Samples should be gently inverted 3-6 times to ensure proper mixing of the blood and anticoagulant. “Gently” is the operative word since vigorous mixing might lead to hemolysis or platelet activation.

In the hospital setting, specimens are often collected from central venous lines. These specimens can lead to partially clotted, clotted, hemolyzed or activated samples. Another great risk with central lines is the likelihood of heparin contamination. Specimen collection from central lines always includes a process for flushing the line and discarding enough blood to protect against said contamination.

Another important pre-analytic consideration is the time until testing is completed. Samples for coagulation tests should be returned to the laboratory as quickly as possible. Some experts say as soon as 1 hour from time of collection, but it is important that the PT and APTT be tested within 4 hours of collection. A PT is stable for analysis for up to 24 hours at room temperature, but the APTT should not be run after four hours of collection.

Sample Processing and Storage

Sample processing and storage are also important when performing coagulation studies. Not all tests are run in all laboratories and care should be taken to provide the reference laboratory with the best sample possible. Most of the common studies, such as PT, APTT and clotting factor assays, are performed on samples that have been centrifuged once. However, lupus anticoagulant assays are performed on samples that have been centrifuged twice prior to freezing to provide the laboratory with platelet-poor plasma. Centrifuge coagulation tubes at less than 1500g for 10-15 minutes to prevent platelet activation. Once separated, plasma should be frozen. Frozen samples at -20øC are stable up to 4 weeks.

Issues that arise from testing usually stem from the pre-analytical phase of the sample. While instruments are often equipped with clot sensors, any prolonged clotting time should cause one to investigate the sample itself. The sample should be inverted and examined visually for obvious clots. If none are seen, insert two wooden applicator sticks to check for clots. Clotted samples are rejected. Other common pre-analytic causes for prolonged clotting times are the presence of EDTA in the sample or heparin contamination. As previously stated, hemolysis, badly processed samples, and lipemia will affect coagulation results. Additionally, specimens with hematocrits over 55% will give errant results because of the improper ratio of anticoagulant to sample.

Time of day is an overlooked factor in coagulation testing. Some components follow circadian rhythm and results will vary at different times of the day. It is important that coagulation studies are drawn the same approximate time each day. Fibrinogen levels are higher in the morning.

Patients who have experienced a clotting event, such as a venous thrombosis or pulmonary embolism, will lose (due to consumption) some of the natural anticoagulants; therefore, care should be taken in determining the presence of a deficiency too quickly.

Kim Ledingham is a medical technologist and freelance writer.

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