Preanalytic Variables in Special Coagulation Testing
Published: October 2008Print Record of Viewing
Proper processing and handling of specimens for coagulation testing is essential to ensure specimen integrity and accurate test results. Dr. Pruthi offers a case-based presentation to discuss the clinical implications of selected preanalytic variables in special coagulation testing and interpretation.
Presenter: Rajiv K Pruthi, M.B.B.S. of the Division of Hematopathology at Mayo Clinic
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Proper processing and handling of specimens for coagulation testing is essential to ensure specimen integrity and accurate test results. Dr. Pruthi uses a case-based presentation to discuss the clinical implications of selected preanalytic variables in special coagulation testing and interpretation.
Variables Affecting Test Results
There are multiple variables that affect coagulation test results, which can be broadly classified into preanalytical or analytical variables.
Preanalytic Variables Affecting Coagulation Testing
Selected preanalytical variables include specimen lipemia, patient hematocrit, collection volume, type of anticoagulant, and specimen processing and storage.
Special Coagulation Test Algorithms
Commonly ordered coagulation test include prothrombin time (PT) and activated partial thromboplastin time (aPTT). Additional specialized assays include individual clotting factor assays, and assays for anticoagulant proteins such as protein C and antithrombin.
DNA-based on molecular tests are not based on the clotting assays, such as the PT and aPTT, and have a different set of preanalytical variables affecting results.
In order to illustrate the impact on selected preanalytical variables on patient management, we will show two illustrated cases.
In this case, a 47-year-old male, with a complex cyanotic congential heart disease, which consisted of a single ventricle with transposition of great vessels and severe pulmonary hypertension, was seeing his cardiologist for a follow-up.
His cardiologist was planning a cardiac angiogram. Given the patient’s cyanotic congenital heart disease his hematocrit was markedly elevated as shown.
Preprocedure Coagulation Tests
The cardiologist had ordered a pre-procedure PT and aPTT, which to be both noted to be moderately prolonged.
PT and aPTT Diagram
This next slide provides a framework to understand the relationship between the coagulation cascade and screening tests such as the PT and the aPTT. The coagulation cascade consists of extrinsic and intrinsic pathways which converge on a final common pathway.
Performance of the PT consists of adding either recombinant tissue factor or other sources of the tissue from the plastin to patient’s plasma along with calcium and phospholipids. This results in activation of factor 7 in the extrinsic pathway, which leads to activation of components of the final common pathway, and ultimately clot formation. The prothrombin time is the time that it takes for the clot to form.
Likewise, for the aPTT test, a contact activator, calcium and phospholipid, are added to patient plasma and the time to clot represents the aPTT. So, a prolonged AT or aPTT may be due to coagulation facet deficiencies or inhibitors of one or more of the coagulation pathways. The aPTT is typically prolonged by anticoagulants like heparin and direct thrombin inhibitors.
Normal Hematocrit and High Hematocrit
This slide shows a cartoon of a specimen collection tube containing citrate anticoagulant. This is just a diagrammatic representation.
When a patient sample is collected the citrate anticoagulant will typically be mixed in and not visible as a discrete layer. The amount of citrate anticoagulant present in commercially available collection tubes is calculated to result in a ratio of 1 part anticoagulant to 9 parts whole blood or 1 part anticoagulant to 5 parts of plasma, as shown.
The tube shown on the left represents a typical blood sample from a patient with a normal hematocrit. The tube on the right, however, represents a sample from a patient with an elevated hematocrit, such as the patient I have just described.
In this situation, even the increase hematocrit there is a relative reduction in the amount plasma collected in the tube, and therefore, there is a relative excess of anticoagulant which elates an excessive amount of calcium.
Preprocedure Coagulation Tests
So when that sample is analyzed, the amount of added assay or calcium is insufficient to activate the coagulation cascade, thus resulting in an artifactually prolonged PT and our aPTT.
To get an accurate result based on published guidelines, the volume of citrate anticoagulant was reduced to 0.25 percent to accommodate the lower volume of plasma. The assays were repeated and indeed were near-normal or normal.
In essence, this patient had an artifactual prolongation of the PT and aPTT due to increased hematocrit. This is not a commonly recognized phenomenon, patient with disease such as cyanotic congenital heart disease and polycythemia rubra vera should be flagged so that specimens can be collected in tubes with adjusted citrate concentration.
The next case is one of a 45 year-old male referred to our hemophilia center for evaluation of diagnosis of mild congenital hemophilia. He had undergone a dental extraction and had more then expected hemorrhage lasting for about a week.
He was tested locally, where von Willebrand factor antigen and ristocetin co-factor activity when noted to be normal; however, Factor VIII was mildly reduced. He brought in his outside medical records and next to the Factor VII results there was an asterisk indicating that a clot was noted in the specimen tube.
Repeat Testing With Fresh Sample
When we repeated his Factor VIII activity using a fresh sample, the Factor VIII was noted then to be normal. A detailed clinical evaluation revealed that his post dental extraction bleeding was felt to be due to consumption of excessive amounts of ibuprofen which he was taking for pain due to the dental extraction. Ibuprofen induces a platelet dysfunction.
Presents of clot formation in the specimen tube had resulted in a consumption of Factor VII resulting in a misdiagnosis of mild hemophilia A. In addition to such consumption, Factor VIII and Factor V are particularly sensitive to the freeze-thaw effect and may sometimes result in a loss of 10-20% activity.
Intrinsic and Extrinsic Diagram
As you can see from this diagram, fibrin clot formation requires activation of clotting factors within the intrinsic, extrinsic, and final common pathways, thus, if assayed, clotting factors in a clotting tube will be reduced.
Clotted Specimen: Definition of Clotted Specimen
A question commonly asked is “How large does the clot need to be in order for such artifactual results to occur?” There are no validation studies looking at the size of a clot within a tube and its affect on clotting factor assays. So for practical purposes a clot found within a specimen tube should be considered as a clotted specimen.
Significance of Clotted Specimen
As I indicated before, in addition to containing clotting factors, plasma also contains the anticoagulant factors. So, the clotting process consumes clotting factors to form the fibrin clot. This results in prolongation of clotting times such as the PT and aPTT, and if assayed results in reduced clotting factors.
In addition the anticoagulant proteins such as protein C, antithrombin and protein S also get consumed by the clotting process. So, if these proteins are assayed from a clotted specimen there is potential for a misdiagnosis of an anticoagulant deficiency state which impacts long-term anticoagulant patient with thrombosis.
Preanalytic Factors Affecting Testing Results: Clotted Specimen
It is well-established that proper specimen collection and processing and timing of testing are all critical steps in reducing the risk of clotted specimens
Scope of the Problem
In the Mayo Clinic Special Coagulation Laboratory we wanted to investigate how often we encountered clotted specimens. As in any large laboratory it is important to understand the scope of the problem in order to be able to institute continuous improvement processes and then to be able to measure outcomes of changes in processes.
Event Capture Tool
As part of a department-wide quality system essential, the Special Coagulation Laboratory included instituted and event capture tool. We rigorously collected data on clotted specimens in order to understand the scope of the problem.
Clotted Specimens as a Percentage of Test Volume
Over a period of about one year, data was collected and analyzed. As shown, between 0.4 and 0.7 percent of all our specimens were clotted. This might not seem like a large number until one looks at the data in absolute terms.
Clotted Specimens 2007
When looking at the data in absolute terms, over a period of about one year, on a monthly basis, between 80 and 100 specimens were found to be clotted. Now that we know the scope of the problem, one wonders “what are the implications for patients.”
Clinical Laboratory Standards Institute
Based on CLS guidelines, clotted specimens received for coagulation testing should be rejected.
Rejection of Specimen: Impact
Rejection of patient specimens as wide ranging impact including: the ordering physician, laboratory technologists and the performing laboratory. Most of all, it affects patient, who may already be worried because of their ongoing illness, who is now informed by their physician’s office that they would have to come back for another blood draw. This may mean time away from work, costs associated with travel to the laboratory, and most likely an addition phlebotomy charge. So based on our data between 80 and 160 patients being served by Mayo Medical Laboratory clients may also be affected on a monthly basis. This is likely not sustainable, which is what prompted us further.
Causes of Clotted Specimen
We attempted to determine potential patient variables that may influence clotted specimens, so we looked at the type of test that were ordered on the specimens that were found to be clotted. As an example, we tried to determine the type of test ordered, by that I mean we looked into whether these clotted samples had thrombosis profiles or components; or bleeding profiles or components ordered. We found no trend toward one type of order or another, making individual patient variables less likely.
It is well-established that optimal sample collection and processing is key to avoiding clotted specimens, however; this would be a daunting study to do given the numerous client laboratories and technologists involved.
Steps Towards Reducing Clotted Specimens
Given the complexities and the scope of this problem, we elected to focus on raising an awareness of the scope of the problem with this current presentation and providing a concentrated-focus education geared to its optimal specimen collection and processing.
This consists of a technical presentation by the Special Coagulation Laboratory assistant supervisor Joel Cayou and a detailed write up in our monthly Communiqué.
In conclusion, I hope that I have been able to highlight the importance of preanalytic variables that affect results of coagulation testing and have been able to emphasize the importance of the specimen collection, processing and transport as being major factors among multiple variables.
Hopefully with this education effort, we may be able to reduce the number of clotted specimen events that will in the end benefit the most important person, the patient. Thank you for your attention.