Background
Disorders of blood clotting include familial diseases such as Hemophilia to the very rare Antithrombin III deficiency. Coagulation disorders are much more common than is clinically realized. Traditionally hematologists have been consulted for many of these problems. The pathologist is often overlooked in obtaining consultation for these disorders. A hematopathologist or a transfusion medicine specialist, both subspecialties of pathology, can be a valuable resource. The link to the Web Coag by Dr. Nguyen (see internet links) is highly recommended as a resource created by a hematopathologist.
- Anti-Phospholipid Antibody
- Blood Banking and Transfusion Medicine
- Coagulation Overview-Web Coag (Dr. Andy N.D. Nguyen of University of Texas Health Science Center)
- Heparin Induced Thrombocytopenia
- Idiopathic Thrombocytopenic Purpura (Autoimmune Thrombocytopenic Purpura, ITP)
- von Willebrand Disease
CLINICAL VARIANTS CHARACTERIZATION Adapated from Ann Diagn Pathol 2001;5:177-183 INHERITED Activated Protein C resistance due to factor V Leiden mutation
Increased factor VIII activity
Antithrombin deficiency
Protein C deficiency
Protein S deficiency
Prothrombing 20210 gene mutationACQUIRED Pregnancy (especially pospartum)
Advanced age
Estrogen use
Immobilization
Trauma
Postoperative state
Antiphospholipid syndrome
Hepain-induced thrombocytopeniaHYPERHOMOCYSTEINEMIA OUTLINE
Laboratory/Radiologic/Other Diagnostic Testing Gross Appearance and Clinical Variants Prognosis Treatment Commonly Used Terms Internet Links
DISEASE VARIANTS AND ASSOCIATIONS CHARACTERIZATION ACTIVATED PROTEIN C RESISTANCE Diagnostic Single Nucleotide Polymorphism Analysis of Factor V Leiden and Prothrombin 20210G>A
A Comparison of the Nanogen Electronic Microarray With Restriction Enzyme Digestion and the Roche LightCycler
Iris Schrijver, MD
Marla J. Lay
and James L. Zehnder, MDAm J Clin Pathol 2003;119:490-496 Abstract quote
Genetic thrombosis risk factors include a sequence variant in the prothrombin gene (20210G>A) and factor V Leiden (1691G>A). These single nucleotide polymorphisms can be diagnosed with restriction fragment length polymorphism (RFLP) analysis, fluorescent genotyping on the LightCycler (Roche Diagnostics, Indianapolis, IN), and microarray-based testing on the novel NanoChip electronic microarray (NanoChip Molecular Biology Workstation, Nanogen, San Diego, CA).We compared these methods for accuracy, time to results, throughput, and interpretation. Results from 789 of 800 individual amplicons analyzed on the NanoChip were in complete agreement with the other assays. Eleven were "no calls" (uninterpreted by the NanoChip system) resulting from failed polymerase chain reaction amplifications. Although the NanoChip System, when used in a low-throughput setting, requires more overall time than the LightCycler, it is nearly equivalent per genotyping call.
Owing to minimal sample handling, assay results are more reliable on the NanoChip platform and on the LightCycler than with RFLP. The NanoChip assay is reliable and may be especially valuable to laboratories with a large volume of thrombophilia test requests.
Activated protein C resistance, the factor v leiden mutation, and a laboratory testing algorithm.Van Cott EM, Soderberg BL, Laposata M.
Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston.
Arch Pathol Lab Med 2002 May;126(5):577-82 Abstract quote Objectives.-To present the current understanding of factor V Leiden and activated protein C resistance, and to propose a laboratory testing algorithm.
Data Sources.-Publications on MEDLINE with the terms factor V Leiden or activated protein C resistance through mid 2001, as well as publications in the authors' files, were screened for inclusion in this report.
Study Selection.-Original studies that report a novel finding on testing or clinical features of activated protein C resistance or factor V Leiden are included.
Data Extraction.-The novel or key findings from the selected studies are analyzed.
Data Synthesis.-Protein C and protein S are the integral components of an anticoagulation pathway that limits fibrinogen conversion to fibrin through the degradation of factors Va and VIIIa. When factor Va is resistant to degradation by activated protein C, this anticoagulation pathway does not operate properly, and patients have an increased risk for thrombosis. This report describes the protein C/protein S pathway, the significance of activated protein C resistance and the factor V Leiden mutation, and the clinical testing used to detect activated protein C resistance and the factor V Leiden mutation. A proposed laboratory testing algorithm is also provided.
Conclusions.-Factor V Leiden is a risk factor for venous thrombosis and it is particularly common in white populations. A laboratory testing algorithm is proposed.
Detection of factor v leiden and prothrombin gene mutations in patients who died with thrombotic events.Ranguelov RD, Rosenthal N, Bromley C, Vasef MA.
Department of Pathology, University of Iowa, Iowa City.
Arch Pathol Lab Med 2002 Oct;126(10):1193-6 Abstract quote Context.-Individuals with factor V or prothrombin gene mutations are at increased risk for thrombotic events. Furthermore, the risk of recurrent deep venous thrombosis in heterozygous carriers of both factor V Leiden and prothrombin gene mutations is high enough that some investigators suggest lifelong warfarin prophylaxis for these individuals, even with a single spontaneous thrombotic event.
Objectives.-To assess the incidence of factor V Leiden and prothrombin gene mutations in an autopsy population and to determine if these tests can prove useful in identification of at-risk family members.
Design.-We analyzed factor V Leiden and prothrombin gene mutations in 45 patients who died with or of thrombotic events, using archival tissue and multiplex allele-specific polymerase chain reaction amplification. The wild-type factor V gene was amplified in all 45 patients, whereas the wild-type prothrombin gene was amplified in 29 patients.
Results.-Two patients (4.4%) who died with thrombotic events at the ages of 35 and 92 years were heterozygous for factor V gene mutation. Two additional patients (6.7%), who died with thrombotic events at the ages of 26 and 39 years, were heterozygous for prothrombin gene mutation. Patients homozygous for either factor V or prothrombin gene or simultaneously heterozygous for both genes were not detected in our study.
Conclusions.-Our findings suggest that screening the relatives of elderly patients who die with thrombotic events would not be cost-effective because of the low incidence of these mutations in the autopsy population. However, because the incidence of these mutations appeared significantly more frequently among individuals who died at 39 years or younger, testing the relatives of this subset of patients may prove useful for detection of at-risk individuals who would benefit from preventive anticoagulation therapy.
ASPIRIN RESISTANCE TESTING
- Narrative review: aspirin resistance and its clinical implications.
Sanderson S, Emery J, Baglin T, Kinmonth AL.
University of Cambridge, Addenbrooke's Hospital, and Institute of Public Health, Cambridge, United Kingdom.
Ann Intern Med. 2005 Mar 1;142(5):370-80. Abstract quote
Aspirin is currently the most cost-effective drug for the secondary prevention of cardiovascular disease, but treatment failures are relatively common. Several factors have been linked to these recurrent vascular events in patients prescribed aspirin, including smoking, drug interactions, nonadherence, comorbid conditions, and aspirin resistance.
The term aspirin resistance has been used to describe not only an absence of the expected pharmacologic effects of aspirin on platelets but also poor clinical outcomes, such as recurrent vascular events, in patients treated with aspirin. Aspirin resistance is perhaps more precisely understood as the phenomenon of measurable, persisting platelet activation that occurs in patients prescribed a therapeutic dose of aspirin and may underlie an unknown proportion of aspirin treatment failures.
Key challenges for future research are to standardize a definition of aspirin resistance and to compare whether different measures of platelet activation, either alone or in combination, independently predict cardiovascular events. These challenges must be met before researchers conduct studies to assess the clinical utility of testing on patient outcomes and cost-effective prescribing.
- Lack of aspirin effect: aspirin resistance or resistance to taking aspirin?
Cotter G, Shemesh E, Zehavi M, Dinur I, Rudnick A, Milo O, Vered Z, Krakover R, Kaluski E, Kornberg A.
Clinical Pharmacological Research Unit, The Cardiology Institute, Zerifin, Israel.
Am Heart J. 2004 Feb;147(2):293-300. Abstract quote
BACKGROUND: A lack of aspirin effect on platelets after a myocardial infarction (MI) is associated with poor health outcome. This lack of effect may be due to biological resistance to aspirin or due to nonadherence (the patient is not taking the aspirin, hence it has no effect). Determining which of these factors predicts poor outcome would inform potential intervention strategies.
METHODS: Aspirin effect on platelets was assessed in a cohort of MI survivors who were divided into three groups: group A ("adherent"), patients whose platelets were affected by aspirin; group B ("nonadherent"), patients whose platelets showed no aspirin effect and who admitted in an interview that they were not taking their medications; and group C (potentially biologically resistant to aspirin), patients whose platelets showed no aspirin effect but maintained that they were taking their aspirin. Two health outcome measures (death, reinfarction, or rehospitalization for unstable angina; or admission for any cardiovascular causes) were assessed 12 months after enrollment.
RESULTS: Seventy-three patients were enrolled and classified into groups A ("adherent," 52 patients), B ("nonadherent," 12 patients), and C ("potentially aspirin resistant," 9 patients). Adverse events and readmission were more common in the nonadherent group (B)-42% and 67%, respectively, when compared with the adherent group (A)-6% and 11%, and with the potentially biologically resistant group (C)-11% and 11%.
CONCLUSIONS: Nonadherence is a significant mediator of poor outcome. It is important to evaluate whether or not patients are taking their medications in clinical settings and in studies that evaluate the effect of prescribed medications.FACTOR VIII Comparison of Three Methods for Measuring Factor VIII Levels in Plasma
Wayne L. Chandler, MD, Chris Ferrell, MT(ASCP), Joo Lee, MT(ASCP), Theingi Tun, MT(ASCP), and Hien Kha, MT(ASCP)Am J Clin Pathol 2003;120:34-39 Abstract quote
We compared 1-stage clot-based, chromogenic, and immunoassay methods for measuring factor VIII in plasma with a focus on the measurement of elevated levels of factor VIII. The chromogenic assay showed the best interassay imprecision for factor VIII levels near 150 IU/dL.
The best correlation was between the 1-stage clot-based and chromogenic factor VIII assays (r2 = 0.934), and the lowest correlation was between the 1-stage clot-based and antigenic factor VIII assays (r2 = 0.821). The presence of heparin, low-molecular-weight heparin, lepirudin, or lupus inhibitors in the sample resulted in major interference in the 1-stage clot-based assay but not the chromogenic or antigenic factor VIII assays.
Overall, the chromogenic factor VIII activity assay was the optimal method, showing good precision, the best overall correlation with other assays, and no interference from heparin, low-molecular-weight heparin, lepirudin, or lupus inhibitors.
PROGNOSIS AND TREATMENT CHARACTERIZATION PROGNOSIS Association Between CYP2C9 Genetic Variants and Anticoagulation-Related Outcomes During Warfarin Therapy
Mitchell K. Higashi, PhD; David L. Veenstra, PharmD, PhD; L. Midori Kondo, PharmD; Ann K. Wittkowsky, PharmD; Sengkeo L. Srinouanprachanh, BS; Fred M. Farin, MD; Allan E. Rettie, PhD
JAMA. 2002;287:1690-1698 Abstract quote
Context
Warfarin is a commonly used anticoagulant that requires careful clinical management to balance the risks of overanticoagulation and bleeding with those of underanticoagulation and clotting. The principal enzyme involved in warfarin metabolism is CYP2C9, and 2 relatively common variant forms with reduced activity have been identified, CYP2C9*2 and CYP2C9*3. Patients with these genetic variants have been shown to require lower maintenance doses of warfarin, but a direct association between CYP2C9 genotype and anticoagulation status or bleeding risk has not been established.Objective
To determine if CYP2C9*2 and CYP2C9*3 variants are associated with overanticoagulation and bleeding events during warfarin therapy.Design and Setting
Retrospective cohort study conducted at 2 anticoagulation clinics based in Seattle, Wash.Participants
Two hundred patients receiving long-term warfarin therapy for various indications during April 3, 1990, to May 31, 2001. Only patients with a complete history of warfarin exposure were included.Main Outcome Measures
Anticoagulation status, measured by time to therapeutic international normalized ratio (INR), rate of above-range INRs, and time to stable warfarin dosing; and time to serious or life-threatening bleeding events.Results
Among 185 patients with analyzable data, 58 (31.4%) had at least 1 variant CYP2C9 allele and 127 (68.6%) had the wild-type (*1/*1) genotype. Mean maintenance dose varied significantly among the 6 genotype groups (*1/*1 [n = 127], *1/*2 [n = 28], *1/*3 [n = 18], *2/*2 [n = 4], *2/*3 [n = 3], *3/*3 [n = 5]) (by Kruskall-Wallis test, 25 = 37.348; P<.001). Compared with patients with the wild-type genotype, patients with at least 1 variant allele had an increased risk of above-range INRs (hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.03-1.90). The variant group also required more time to achieve stable dosing (HR, 0.65; 95% CI, 0.45-0.94), with a median difference of 95 days (P = .004). In addition, although numbers were small for some genotypes, representing potentially unstable estimates, patients with a variant genotype had a significantly increased risk of a serious or life-threatening bleeding event (HR, 2.39; 95% CI, 1.18-4.86).Conclusions
The results of our study suggest that the CYP2C9*2 and CYP2C9*3 polymorphisms are associated with an increased risk of overanticoagulation and of bleeding events among patients in a warfarin anticoagulation clinic setting, although small numbers in some cases would suggest the need for caution in interpretation. Screening for CYP2C9 variants may allow clinicians to develop dosing protocols and surveillance techniques to reduce the risk of adverse drug reactions in patients receiving warfarin.
TREATMENT CHARACTERIZATION CRYOPRECIPITATE Cryoprecipitate
Patterns of Use
Liron Pantanowitz, MD, Margot S. Kruskall, MD, and Lynne Uhl, MD
Am J Clin Pathol 2003;119;874-881 Abstract quote
The type of coagulation factors and proteins in cryoprecipitate determine the appropriate indications for its use. To determine the pattern of use at a tertiary care medical center, we performed a retrospective audit of cryoprecipitate utilization. A total of 51 patients received 88 pools of cryoprecipitate.
In 39 patients, cryoprecipitate was transfused for appropriate indications: hypofibrinogenemia (n = 19), tissue plasminogen activator reversal (n = 1), management of massive transfusion (n = 7), correction of uremic bleeding (n = 2), and for making fibrin sealant (n = 10). Overall, these patients used approximately 80% of the cryoprecipitate transfused. In 12 other patients, cryoprecipitate was transfused inappropriately to attempt reversal of the anticoagulant effects of warfarin therapy (n = 6), to treat impaired surgical hemostasis in the absence of hypofibrinogenemia (n = 4), and to treat hepatic coagulopathy with multiple factor deficiencies (n = 2).
The patterns of misuse, involving 24% of all cryoprecipitate orders, suggest a widespread misunderstanding and need for focused education about the coagulation factors and proteins present in cryoprecipitate and appropriate indications for its use.REFRACTORY PLATELET TRANSFUSION
Management of patients refractory to platelet transfusion.Sacher RA, Kickler TS, Schiffer CA, Sherman LA, Bracey AW, Shulman IA; College of American Pathologists.Transfusion Medicine Resource Committee.
University of Cincinnati Medical Center, Hoxworth Blood Center, Cincinnati, Ohio, USA
Arch Pathol Lab Med 2003 Apr;127(4):409-14 Abstract quote OBJECTIVE: To present a current assessment and practical approach to the diagnosis and management of patients who are refractory to platelet transfusions.
DESIGN: A task force was convened by the College of American Pathologists under the auspices of the Transfusion Medicine Resource Committee for the purposes of outlining current concepts in the definition and diagnosis of this difficult clinical management problem and selection of the optimal platelet component for these patients.
RESULTS: This article represents a contemporary approach to the diagnosis and management of patients who are refractory to platelet transfusions. This document is based on a current literature review and dialog among members of the task force convened to address the subject.
CONCLUSIONS: It is hoped that this document will represent a resource and practical approach to the issue of diagnosis and management of patients who are refractory to platelet transfusions.
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