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Pre-analytical variables in coagulation testing

Pre-analytical phase includes all processes involved in obtaining, collecting, transporting, and preparing samples prior to analysis. This phase represents a major source of laboratory errors. Prior to testing, a bleeding history is a prerequisite for the diagnosis of any bleeding disorder and should guide further laboratory investigations. It is important to know the manner and age of appearance of hemorrhages, their frequency, and whether bleeding is aggravated after taking some medication. Any interfering drugs should be administered after collecting the blood sample. A record of family history of bleeding and details of all the drugs that the patient had taken during the week prior to testing should be taken. Blood samples should preferably be collected from fasting subjects, who have refrained from smoking/consumption of caffeine/physical activity, 2 hours prior to sampling. Laboratory tests for hemostasis require citrated plasma collected in tubes containing 3.2 percent sodium citrate in a ratio of 9 parts blood and 1 part anticoagulant. This is important because PT and aPTT tests require the addition of calcium. If the specimen contains excess citrate, addition of calcium may be inadequate, and the low-plasma calcium will lead to an artificial prolongation of PT or aPTT.

Under-filling of the tube may also result in underestimation of the clotting factor levels. Blood should be collected in an atraumatic way, and application of tourniquet should be restricted to less than 1 minute. During collection, order of draw should be followed – the blood coagulation tube is first when multiple containers are to be collected except when a blood culture is needed. When using a winged blood-collection set for venipuncture and a coagulation tube is the first tube to be drawn, a discard tube should be drawn first as the air volume contained in the tubing partially fills the vacuum tube, leading to insufficient filling of the tube with citrate. Heparinized needles are not to be used as it might falsely elevate PT and aPTT. After collecting the sample, it should be mixed properly in the figure-of-eight fashion. Insufficient mixing may have effect on specialized hemostasis assays performed, and avoid vigorous shaking of the tube as it might lead to in-vitro hemolysis or spurious factor activation, resulting in false shortening of test-clotting times. Pre-analytical issues can also be seen if the patient’s hematocrit is <30 percent or >55 percent. When the hematocrit is abnormal due to either severe anemia or polycythemia, the blood:citrate ratio needs to be adjusted. Centrifugation of the sample should ideally be at 1500 g for a minimum of 10–15 min; plasma sample needs to be frozen in laboratory and transported in dry ice. All blood specimens for coagulation utilize platelet-poor plasma (PPP). PPP is defined as plasma having <10,000 platelets/µL. PPP is required because platelets can interfere with coagulation tests when they rupture or activate. Testing generally requires using the once-centrifuged test sample; however, for some assays, such as lupus anticoagulant, the samples should be double centrifuged, which entails the re-centrifugation of the separated plasma, and re-separation of this double-spun plasma from any residual cellular pellet prior to freezing.

Pre-analytical variables may be associated with the patient themselves, specimen collection, specimen transportation, and specimen processing and storage. These can cause significant changes in values obtained from routine and specialized coagulation testing. Failure to recognize or address these pre-analytical variables may create factitious test values, resulting in patient mismanagement, including misdiagnosis, improper dosing, and improper treatment.

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