Laboratory Evaluation of Thrombophilia.

Venous thromboembolism (VTE) occurs typically in the veins of the lower extremities and/or as pulmonary embolism. There is a myriad of causes of VTE ranging from provoked causes (e.g., surgery, cancer) to unprovoked causes (e.g., inherited abnormalities) or multiple factors that combine to initiate the cause. Thrombophilia is a complex, multi-factorial disease that may result in VTE. The mechanism(s) and causes of thrombophilia are complex and not completely understood. In healthcare today, only some answers about the pathophysiology, diagnosis, and prevention of thrombophilia have been elucidated. The laboratory analysis for thrombophilia is not consistently applied, and has changed over time, but remains varied by providers and laboratories as well. Both groups must establish harmonized guidelines for patient selection and appropriate conditions for analysis of inherited and acquired risk factors. This chapter discusses the pathophysiology of thrombophilia, and evidence-based medicine guidelines discuss the optimum laboratory testing algorithms and protocols for selection and analyzing VTE patients to ensure a cost-effective use of limited resources.

Laboratory Evaluation of Antithrombin, Protein C, and Protein S.

Thrombophilia is a complex disease process, clinically manifesting in various forms of venous thromboembolism. Although both genetic and acquired (or environmental) risks factors have been reported, the presence of a genetic defect (antithrombin [AT], protein C [PC], protein S [PS]) is considered three of the major contributing factors of thrombophilia. The presence of each of these risk factors can be established by clinical laboratory analysis; however, the clinical provider and laboratory personnel must understand the testing limitations and shortcomings associated with the assays for these factors to be able to ensure an accurate diagnosis. This article will describe the major pre-analytical, analytical, and post-analytical issues associated with the various types of assays and discuss evidence-based algorithms for analyzing AT, PC, and PS in plasma.

Cost-Effective Use of the Protein S Algorithm in Thrombophilia Testing.

BACKGROUND: One of the most complex risk factors for the laboratory assessment of thrombophilia is Protein S (PS). The testing algorithm for PS employs the plasma-based assays of free PS antigen, total PS antigen, and PS activity creating a complex diagnostic scheme that can lead to misdiagnosis if incorrectly used, and a potential waste of resources and money. CONTENT: This paper compares the recently published evidence-based algorithm from the International Society for Hemostasis and Thrombosis (ISTH) with several commonly performed nonevidence-based testing schemes, to demonstrate the efficiency of the evidence-based algorithm for diagnostic efficiency with improved patient care and increased cost savings for the laboratory. SUMMARY: Significant savings (31%-60%) can be realized when the evidence-based algorithm is used in place of other testing modalities of initial PS activity testing (31%) or testing with all 3 assays simultaneously (60%). This study utilizing the PS testing evidence-based algorithm as part of a thrombophilia evaluation demonstrates that the appropriate testing methods can be used to limit wasteful practices while achieving the maximum level of information in this time of limited resources and need for increase monetary savings.

Guidance for establishing a factor VIII testing protocol for the myriad of factor VIII products.

INTRODUCTION: Management of hemophilia A has changed significantly in the past few years with the expansion of new and/or modified products as treatment options. Unfortunately, many of the standard factor VIII assays do not always accurately measure all available treatment products; therefore, the laboratory must investigate various assay algorithms to ensure the reporting of the correct results. METHODS: Requirements for factor testing, diagnosis and severity levels, product testing, factor VIII inhibitor detection and titers, are evaluated, and potential algorithms are created for optimal assessment of patients with hemophilia A. RESULTS: The potential for inaccurate result reporting for patients with hemophilia A or those being treated with the myriad of products has left many laboratories uncertain as to which assay algorithm to implement to ensure reporting the correct results for all products used in their hemophilia program. Algorithms for using either One-stage Clotting assays or Chromogenic assays or a combination of both types of assays are presented for each laboratory to implement based on their clinical situation. CONCLUSIONS: Several algorithms are considered based on the needs of the clinical providers and their patients. Each laboratory must select a testing algorithm that is cost-effective and within available resources, yet that encompasses the needs of their providers and patients. Laboratory personnel must consider all assay uses (factor VIII levels, different products, interfering products, and inhibitor titers) in determining the best algorithm for their laboratory. This paper is a starting guide for developing the best factor VIII testing assays and protocols for your laboratory.

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing.

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Guidance on the critical shortage of sodium citrate coagulation tubes for hemostasis testing.

Recent manufacturing problems and increased utilization has created a shortage of 3.2% sodium citrate blood collection tubes used for coagulation testing, causing stakeholders such as hospitals, clinics and laboratories, to find suitable alternatives. Considerations for in-house citrate blood collection tube preparations or purchasing commercial products from unknown manufacturing sources is of particular concern to laboratories that perform coagulation testing. It is well recognized that variability exists between citrate blood collection tube manufacturers, thereby making any transition to new blood collection methods more challenging than simply switching to a new source. This document provides provisional guidance for validating alternative sources of sodium citrate blood collection tubes (commercial or in-house preparations) prior to clinical implementation.

International Council for Standardisation in Haematology (ICSH) recommendations for collection of blood samples for coagulation testing.

This guidance document has been prepared on behalf of the International Council for Standardisation in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for collection of blood samples for coagulation tests in clinical laboratories throughout the world. The following processes will be covered: ordering tests, sample collection tube and anticoagulant, patient preparation, sample collection device, venous stasis before sample collection, order of draw when different sample types need to be collected, sample labelling, blood-to-anticoagulant ratio (tube filling) and influence of haematocrit. The following areas are excluded from this document, but are included in an associated ICSH document addressing processing of samples for coagulation tests in clinical laboratories: sample transport and primary tube sample stability; centrifugation; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport and storage; and preanalytical variables for platelet function testing. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Recommendations for clinical laboratory testing for protein S deficiency: Communication from the SSC committee plasma coagulation inhibitors of the ISTH.

Hereditary deficiencies of protein S (PS) increase the risk of venous thrombosis; however, assessing the plasma levels of PS can be difficult because of its complex physiological interactions in plasma, sample-related preanalytical variables, and numerous acquired disease processes. Reliable laboratory assays are essential for accurate evaluation of PS when diagnosing a congenital deficiency based on the plasma phenotype alone. This report presents the current evidence-based recommendations for clinical PS assays as well as when to test for PS abnormalities.

Pleiotropic anticoagulant functions of protein S, consequences for the clinical laboratory. Communication from the SSC of the ISTH.

Hereditary deficiencies of protein S (PS) increase the risk of thrombosis. However, assessing the plasma levels of PS is complicated by its manifold physiological interactions, while the large inter-individual variability makes it problematic to establish reliable cut-off values. PS has multiple physiological functions, with only two appearing to have significant anticoagulant properties: the activated protein C (APC) and tissue factor pathway inhibitor alpha (TFPIα) cofactor activities. Current clinical laboratory investigations for deficiency in PS function rely only on the APC-dependent activity. This communication presents an argument for reclassifying the qualitative PS deficiencies to differentiate the two major anticoagulant functions of PS. Reliable assays are necessary for accurate evaluation of PS function when making a specific diagnosis of PS deficiency based on the anticoagulant phenotype alone. This report emphasizes the pleiotropic anticoagulant functions of PS and presents evidence-based recommendations for their implementation in the clinical laboratory.

Recommendations for clinical laboratory testing for protein C deficiency, for the subcommittee on plasma coagulation inhibitors of the ISTH.

Inherited protein C (PC) deficiency increases risk of venous thromboembolism (VTE) by 5 to 10-fold in thrombosis-prone families; however, heterozygous PC deficiency alone does not determine that a subject has thrombophilia. Protein C deficient subjects, who lack additional inherited risk factors such as factor V Leiden or have no major acquired risk factors, may not suffer from VTE. In addition, PC deficiency may be acquired, often due to vitamin K antagonist treatment or liver disease. In contrast, homozygous or compound heterozygous PC deficiencies are rare and serious disorders, and affected infants are often in families with no history of PC deficiency or thrombosis. Laboratories commonly use the chromogenic PC assay to diagnose deficiency. Chromogenic assay is recommended due to its good specificity, but this assay fails to detect the rare type 2b deficiency where the defect is due to poor interaction with calcium ions, phospholipid, protein S, and factor Va and factor VIIIa. The clotting-based assay of PC is capable of detecting type 2b deficiency but it has reduced specificity. Importantly, PC level varies with age, adult reference ranges cannot be applied to babies or children and levels may not reach those of adults even in adolescence. Pre-analytical variables in the specimen affect measurement of PC, and can be assay-dependent; for example, a partially clotted sample will have falsely raised PC level by chromogenic assay but reduced level by clotting-based assay. Direct oral anticoagulants falsely raise PC level in the clotting-based assay but the standard chromogenic assay is unaffected.

Clinical utility and impact of the use of the chromogenic vs one-stage factor activity assays in haemophilia A and B.

Treatment of haemophilia A/B patients comprises factor VIII (FVIII) or factor IX (FIX) concentrate replacement therapy, respectively. FVIII and FIX activity levels can be measured in clinical laboratories using one-stage activated partial thromboplastin time (aPTT)-based clotting or two-stage chromogenic factor activity assays. We discuss strengths and limitations of these assays, providing examples of clinical scenarios to highlight some of the challenges associated with their current use for diagnostic and monitoring purposes. Substantial inter-laboratory variability has been reported for one-stage assays when measuring the activity of factor replacement products due to the wide range of currently available aPTT reagents, calibration standards, factor-deficient plasmas, assay conditions and instruments. Chromogenic activity assays may avoid some limitations associated with one-stage assays, but their regulatory status, perceived higher cost, and lack of laboratory expertise may influence their use. Haemophilia management guidelines recommend the differential application of one or both assays for initial diagnosis and disease severity characterisation, post-infusion monitoring and replacement factor potency labelling. Efficient communication between clinical and laboratory staff is crucial to ensure application of the most appropriate assay to each clinical situation, correct interpretation of assay results and, ultimately, accurate diagnosis and optimal and safe treatment of haemophilia A or B patients.

Preanalytical Variables in Coagulation Testing: Setting the Stage for Accurate Results.

Many preanalytical variables may affect the results of routine coagulation assays. While advances in laboratory instrumentation have partially addressed the laboratory's ability to recognize some of these variables, there remains an increased reliance on laboratory personnel to recognize the three potential areas where coagulation testing preanalytical issues may arise: (1) specimen collection (including patient selection), (2) specimen transportation and stability, and (3) specimen processing and storage. The purpose of this article is to identify the preanalytical variables associated with coagulation-related testing and provide laboratory practice recommendations in an effort to improve the quality of coagulation testing and accuracy of result reporting.

Sources and solutions for spurious test results in coagulation.

In the coagulation laboratory, much emphasis has been placed on rapid and accurate testing; however, spurious results that are inaccurate and do not reflect the actual status of the patient can potentially lead to an incorrect diagnosis and altered intervention. Errors in coagulation results and interpretation can occur at any point of the process from obtaining the specimen to interpretation and use of the result by the clinician. The main sources of error include the patient's biological and preanalytical variation, analytical testing, and postanalytical use of the reported result(s). This article reviews various sources of error leading to spurious results, providing methods to recognize these aberrant results and presenting solutions for minimizing their occurrence.

Lack of grading agreement among international hemostasis external quality assessment programs.

: Laboratory quality programs rely on internal quality control and external quality assessment (EQA). EQA programs provide unknown specimens for the laboratory to test. The laboratory's result is compared with other (peer) laboratories performing the same test. EQA programs assign target values using a variety of methods statistical tools and performance assessment of 'pass' or 'fail' is made. EQA provider members of the international organization, external quality assurance in thrombosis and hemostasis, took part in a study to compare outcome of performance analysis using the same data set of laboratory results. Eleven EQA organizations using eight different analytical approaches participated. Data for a normal and prolonged activated partial thromboplastin time (aPTT) and a normal and reduced factor VIII (FVIII) from 218 laboratories were sent to the EQA providers who analyzed the data set using their method of evaluation for aPTT and FVIII, determining the performance for each laboratory record in the data set. Providers also summarized their statistical approach to assignment of target values and laboratory performance. Each laboratory record in the data set was graded pass/fail by all EQA providers for each of the four analytes. There was a lack of agreement of pass/fail grading among EQA programs. Discordance in the grading was 17.9 and 11% of normal and prolonged aPTT results, respectively, and 20.2 and 17.4% of normal and reduced FVIII results, respectively. All EQA programs in this study employed statistical methods compliant with the International Standardization Organization (ISO), ISO 13528, yet the evaluation of laboratory results for all four analytes showed remarkable grading discordance.

Assessment of Hereditary Thrombophilia: Performance of Protein C (PC) Testing.

Protein C (PC) is a plasma Vitamin K-dependent pro-enzyme protein that is synthesized in the liver. Upon activation, PC regulates the coagulation process by neutralizing the procoagulant activities of factors V and VIII in the presence of the cofactor Protein S. PC is a major regulator of the coagulation process. The clotting based Protein C assay, the protocol described in this chapter, quantitates the amount of functional PC present in the specimen in a proportional fashion based on the prolongation of the Activated Partial Thromboplastin Time (APTT). Other methods for assessing PC are also available, including chromogenic and antigenic assays, but protocols for these assays are not provided.

Assessment of Hereditary Thrombophilia: Performance of Protein S (PS) Testing.

Protein S (PS) is a Vitamin K-dependent protein that functions as a cofactor for the regulation of the coagulation system. PS works in conjunction with Activated Protein C to inactivate factors V and VIII. PS circulates in plasma either complexed to the complement protein, C4b Binding Protein or unbound. The unbound (or free) component is the functional form for the regulation of the coagulation system. PS can be measured in plasma by functional activity, the free (or unbound form) or both free and bound fractions (Total PS). The test most widely used for clinical evaluations is the Free PS Antigen assay (which is the surrogate of PS anticoagulant activity) and represents the protocol described in this chapter. The Free PS Antigen assay is an immunologic assay which specifically measures the unbound fraction of PS in test plasma. Other methods for assessing PS are also available, including PS activity and total PS Antigen assays, but protocols for these assays are not provided.

Assessment of Hereditary Thrombophilia: Performance of Antithrombin (AT) Testing.

Antithrombin (AT) is a naturally occurring plasma inhibitor of coagulation, which is a synthesized in the liver. AT inhibits coagulation serine proteases (the enzymatically activated forms of the clotting factors), mainly thrombin (factor IIa) and factor Xa, but also to a lesser extent factors IXa, XIa, and XIIa. Acting alone, AT inhibits coagulation factors, but does this very slowly; however, when coupled with heparin as a cofactor, the speed of inhibition is increased many fold. The AT/Heparin complex is the most powerful naturally occurring anticoagulant in blood. AT levels of <70% of normal can cause significant thrombosis. Low levels of AT are caused by inherited genetic defects or acquired causes from other disease states. Plasma AT levels can be determined using a chromogenic assay with either bovine thrombin or human factor Xa as the enzyme. The generated color generated in the assay is inversely proportional to the concentration of AT in the plasma.

Activated Partial Thromboplastin Time Monitoring of Unfractionated Heparin Therapy: Issues and Recommendations.

When administering unfractionated heparin (UFH), therapeutic levels of anticoagulation must be achieved rapidly and maintained consistently in the therapeutic range. The basic assays for monitoring UFH therapy are the activated partial thromboplastin time (APTT) and/or the chromogenic antifactor Xa or antithrombin assays. For many laboratories, the APTT is the preferred standard of practice; however, the APTT is a surrogate marker that only estimates the heparin concentration. Many factors, including patient variation, reagents of the APTT, UFH composition, and concentration can influence the APTT result. This article reviews various methods to determine the heparin therapeutic range and presents recommendations for the laboratory to establish an APTT heparin therapeutic range for all sizes of hospitals.

Comparison of methods to determine rivaroxaban anti-factor Xa activity.

BACKGROUND AND OBJECTIVES: Rivaroxaban, a new oral anti-Xa agent, has been approved for use without routine monitoring, but the lack of a predictable drug level measurement may hinder the management of anticoagulated patients. The aims of the project were to correlate a Anti-Factor Xa assay using commercial calibrators and controls (Riva Activity) with serum drug levels analyzed by HPLC-MS/MS (Riva MS) in patients currently receiving rivaroxaban, and secondly, to correlate the PT/PTT, thrombin generation (CAT assay) and Thromboelastograph (TEG) with the Riva activity and Riva MS. METHODS: Recruited patients receiving rivaroxaban prospectively had a total of 3 blood samples taken at least 2 hours apart. Plasma was divided for measurement of PT/PTT, Riva activity, rivaroxaban HPCL-MS/MS, and thrombin generation. TEG activity was measured at one random time point for each patient. Correlation and linear regression evaluations were used to compare the different assays. RESULTS: The cases were 22 patients on rivaroxaban, age 56+12.6, and 10 healthy controls. There was a strong correlation between Riva activity compared to serum Riva MS (r=0.99). We found a statistically significant correlation between PT/INR compared to serum measurements of Riva MS (r=0.68) and anti-Xa activity (r=0.69). The peak (r=-0.50) and lag time (r=0.57) CAT correlated with Riva MS measurements. There was no correlation between Riva MS and PTT, TEG R, TEG MA, Endogenous Thrombin potential. CONCLUSION: Riva anti-factor Xa activity assay measured with commercial calibrators and controls provides a reliable assessment of rivaroxaban serum levels for patients requiring measurement of anticoagulant activity. Correlation with other coagulation tests is not sufficiently strong to be used clinically.