Interferences in Coagulation Testing


Vol. 18 • Issue 20 • Page 58

Lab Limelight

The most common coagulation tests are prothrombin (PT) and activated partial thromboplastin time (aPTT). Physicians will often order PT to determine the degree of blood anticoagulation in patients using anticoagulants or blood thinners such as Warfarin as well as to detect acquired bleeding disorders (e.g., vitamin K deficiency and liver damage). PT is more commonly reported in the International Normalized Ratio unit system. aPTT detects hereditary clotting abnormalities and monitors heparin therapy. Both tests are often ordered in conjunction.

Coagulation assays can be affected by pre-analytical process variables such as venipuncture device, type of collection tube, sample transportation delays, storage conditions and volume of blood collected relative to anticoagulant ratios, says Joseph Sweeney, MD, director, Coagulation and Transfusion Services, and professor of Pathology and Laboratory Medicine at Brown University, Providence, RI.

Hemolysis

Hemolysis-the splitting of erythrocytes (red blood cells) and release of their contents (hemoglobin) into surrounding fluid-can occur when blood is collected, particularly from plastic cannulae inserted in patients in emergency rooms, intensive care units or operating rooms. Dr. Sweeney says risk of hemolysis is substantially higher when plastic as opposed to metal cannulae are used, although it’s not fully understood why.

During blood collection, blood travels to a vacutainer tube by a pressure gradient between the vein (which is at atmospheric pressure) and the collection tube (which is at subatmospheric pressure). Red cells are exposed to high shearing forces and travel at high speeds, acquiring kinetic energy, Dr. Sweeney explains. When they hit the bottom wall of the tube, energy is likely converted into heat, damaging the red cell and resulting in formation of small microparticles and leakage of hemoglobin from cytoplasm. When hemoglobin leaks, certain contents of the red cells (e.g., potassium and cytoplasmic enzymes) also leak, which can cause problems with chemistry analyzers.

Coagulation Analyzers

Coagulation analyzers mainly test platelet-poor plasma, distinguished by a straw color if observed with the eye. Hemoglobin, marked by a red hue, stains plasma red when it leaks from red cells. This is sometimes visible when free hemoglobin concentration exceeds 20 mg/dl and nearly always when concentration exceeds 30 mg/dL. Normal free hemoglobin concentration in plasma is <3 mg/dL, so as little as 0.1% hemolysis can cause a visible stain. Most coagulation analyzers use light transmission to detect clots, meaning interference can hamper clot detection. Dr. Sweeney says, “If no clot’s detected, essentially no result is obtained, so the test is a non-event.”

Clinicians use coagulation assays to ascertain whether a patient is adequately but not excessively anticoagulated, the latter of which would put them at risk of bleeding. Bleeding is the major side effect of heparin therapy and can be associated with high morbidity (requiring blood transfusion) or even mortality if bleeding is near the head, Dr. Sweeney says. “It’s not something to be dismissed as being immaterial,” he says.

Rejecting Specimens

In a 2006 American Society for Clinical Pathology article, “The Effect of Specimen Hemolysis on Coagulation Test Results,” authors discuss the Clinical and Laboratory Standards Institute’s PT and aPTT testing guidelines advocating that samples with visible hemolysis should not be used because of possible clotting factor activation and interference with accurate endpoint measurement. Dr. Sweeney, a co-author, says the study revealed that hemolyzed specimen test results are altered only slightly, if at all, by hemolysis.

“The problem about conservative approaches is they give rise to a lot of inefficiencies and logistic difficulties and repeat venipunctures on patients,” he says. “So the guidelines, while they are understandable in the context in which they were developed, are not all that well-supported by scientific data, and therefore it’s up to each lab to examine its own analyzer and see if hemolysis is associated with any artifactural changes.”

If a specimen is hemolyzed at Dr. Sweeney’s lab, technologists add a comment to the report saying the result may be affected by a margin of 10% and that they recommend re-collection if this difference would affect diagnostic or therapeutic decisions. “For many clinicians, that degree of difference in the test result is not clinically material,” Dr. Sweeney says.

Interference Prevention

Developments in preventing coagulation testing interference include analyzers that use optical detection at multiple wavelengths and electromechanical methods such as metallic balls rotating in a magnetic field to detect blood clots. These methods are less likely to be affected by hemolysis or high levels of lipids or bilirubin, which may occur in patients with liver disease, Dr. Sweeney says.

Jill Hoffman is a senior associate editor.

Diagnostica Stago Inc.

The STA Satellite® fully automated benchtop analyzer from Diagnostica Stago Inc. can simultaneously perform clotting, chromogenic and immunologic assays for the low-volume coagulation lab and is ideal for satellite labs. The STA Satellite® provides reliability, operational ease, flexibility and security within a small footprint. Features include an LCD integrated flat screen, integrated barcode reader, bi-directional (ASTM protocol) connection, removable sample carousel (for 20 primary tubes), 80 user-definable test methodologies, ISO 9001 and 13485, CE mark, etc.

Instrumentation Laboratory Company

The Instrumentation Laboratory Company ACL TOP Family of Hemostasis Testing Systems, distributed in North America by Beckman Coulter Inc., offers speed, simplicity and intelligence to meet the varied volume and testing needs of hospitals and specialty labs. Available in a variety of models, ACL TOP systems run routine/specialty assays and operate continuously with real-time stat capability to deliver automated reagent management, quality control and maintenance. They are designed to reduce workload, expand productivity and enable standardization throughout the lab and hospital.

Siemens Healthcare Diagnostics

The BCS® XP system from Siemens Healthcare Diagnostics consolidates workflow for hemostasis testing and offers a user-defined specialty menu. The system maximizes high-volume routine workloads with random-access and walk-away ability for normal and pathological samples. Routine and specialty testing (including clotting, chromogenic, immunologic and agglutination coagulation assays) are available on one platform. Other features include a high dilution range (up to 1:1200), automatic reagent level monitoring, IDM, onboard QC; and up to 380 simultaneous stat, specialty and routine tests per hour.

About The Author