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 mutation
ACQUIRED	Pregnancy (especially pospartum) Advanced age Estrogen use Immobilization Trauma Postoperative state Antiphospholipid syndrome Hepain-induced thrombocytopenia
HYPERHOMOCYSTEINEMIA

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, MD	Am 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.

 

LABORATORY/ RADIOLOGIC/ OTHER	CHARACTERIZATION
GENERAL
In-house calibration of the international sensitivity index or calibration curve for determination of the international normalized ratio. Brien WF, Crawford L, Raby A, Richardson H. University Health Network, Department of Laboratory Hematology, Toronto, Ontario.	Arch Pathol Lab Med. 2004 Mar;128(3):308-12. Abstract quote   CONTEXT: The international normalized ratio (INR) has been used since 1983 to standardize prothrombin time results for patients on oral anticoagulants. However, significant interlaboratory variations have been noted. Attempts have been made to address these differences with the use of instrument-specific International Sensitivity Index (ISI) values and in-house calibration of ISI values. OBJECTIVE: To assess the performance of laboratories using a calibration curve for INR testing. DESIGN: Attempts to improve performance of the INR include the use of instrument-specific ISI values, model-specific ISI values, in-house calibration of ISI values, and more recently, the preparation of a calibration curve. Several studies have shown an improvement in performance using these procedures. In this study of licensed laboratories performing routine coagulation testing in the Province of Ontario, Canada, the determination of the INR by a calibration curve was compared with the laboratories' usual method of assessment. These methods were subsequently analyzed by comparing the results to instrument-specific ISI, model-specific ISI, and in-house calibrators. International normalized ratios derived by both methods were analyzed for accuracy and precision. The stability of a calibration curve was also investigated. RESULTS: Performance of INR testing has improved with use of a calibration curve or in-house calibrators. CONCLUSION: The results confirm that either in-house calibrators or the calibration curve improve performance of INR testing. The calibration curve may be easier to use and appears stable up to 4 months.
Minimum Numbers of Fresh Whole Blood and Plasma Samples From Patients and Healthy Subjects for ISI Calibration of CoaguChek and RapidPointCoag Monitors Leon Poller, DSc, Michelle Keown, MSc, Nikhil Chauhan, PhD, Anton M.H.P. van den Besselaar, PhD, Armando Tripodi, PhD, Jorgen Jespersen, DSc, and Caroline Shiach, MD	Am J Clin Pathol 2002;117:892-899 Abstract quote The international sensitivity index (ISI) calibration of point-of-care-test (POCT) prothrombin time (PT) whole blood monitors is complex, requiring manual PT testing of 60 patients' and 20 healthy subjects' plasma samples. The possibility of reducing these numbers was studied by a Monte Carlo Bootstrap study for 2 POCT PT systems. For reduced sample numbers, this consisted of 50,000 calibrations using whole blood and plasma samples tested on the monitors with manual PT testing of plasma samples from the same blood donations. There was little effect on mean ISI by reduction of sample numbers to a total of 7, but there was progressively less certainty regarding the reliability of the calibration. Precision of the calibrations and international normalized ratio deviation were not affected markedly by reducing numbers to half. As ISI calibration with the 2 POCT systems was less precise than conventional manual testing, for maximum confidence, reduction of numbers is not advised.
FACTOR V LEIDEN POLYMORPHISMS
Activated protein C resistance assay detects thrombotic risk factors other than factor V Leiden. Graf LL, Welsh CH, Qamar Z, Marlar RA. Pathology Research Laboratory, Research Service, Denver Veterans Affairs Medical Center, 1055 Clermont St, Denver, CO 80220, USA.	Am J Clin Pathol 2003 Jan;119(1):52-60 Abstract quote Activated protein C (APC) resistance is a common risk factor for venous thromboembolism (VTE) attributed to various mechanisms, including factor V Leiden (FVL) polymorphism. FVL is considered responsible for up to 95% of APC resistance; however, other factor V polymorphisms and elevated factor VIII levels also have been implicated. We assessed whether additional mechanisms contribute to APC resistance in a blinded case-control study of 65 subjects by measuring APC resistance using 3 methods: 2 activated partial thromboplastin time-based methods with and without dilution in factor V-deficient plasma and 1 Russell viper venom-based assay (RVV). Without factor V-deficient plasma, 24 subjects were APC resistant; with factor V-deficient plasma, the assay identified fewer APC-resistant subjects, as did RVV. All assays detected the 7 heterozygous FVL subjects. Thirteen subjects had factor VIII levels above 150% (1.50). After excluding subjects with FVL or elevated factor VIII levels, 4 subjects still had APC resistance. VTE risk trended higher for subjects with APC resistance in the absence of FVL. Measurement of APC resistance without dilution in factor V-deficient plasma is needed to assess for potentially important thrombotic risk factors other than FVL.
HEPARIN MONITORING
Heparin monitoring and patient safety: a College of American Pathologists Q-Probes study of 3431 patients at 140 institutions. Valenstein PN, Walsh MK, Meier F; College of American Pathologists. Department of Pathology, St Joseph Mercy Hospital, Ann Arbor, Mich 48106-0995, USA.	Arch Pathol Lab Med. 2004 Apr;128(4):397-402. Abstract quote CONTEXT: Appropriate laboratory monitoring of unfractionated heparin therapy promotes effective anticoagulation while minimizing hemorrhagic complications. OBJECTIVES: To measure heparin therapy monitoring in a "real-world" setting and to assess the degree of anticoagulation achieved. DESIGN: One hundred forty institutions abstracted laboratory and pharmacy data from up to 30 inpatients receiving standard-dose unfractionated heparin therapy for 72 hours. Institutions also reported their therapeutic ranges and described heparin prescribing and monitoring policies. RESULTS: Activated partial thromboplastin times or anti-factor Xa levels were measured at least once within the first 12 hours of administration for 95% of 3431 heparinized inpatients. Eighty-seven percent of patients had a platelet count performed within 72 hours of heparin administration. Seventy-eight percent of heparinized inpatients achieved therapeutic anticoagulation within 24 hours, but more than one third of patients entered the supratherapeutic range on at least 2 occasions during the first 72 hours. We found moderate variation in performance among the 140 institutions participating in the study, with more consistency in monitoring patients but less consistency in achieving therapeutic levels of anticoagulation. In one fourth of hospitals, more than half of the heparinized patients entered the supratherapeutic range on 2 or more occasions during the first 72 hours of therapy. None of 20 institutional practices we examined were meaningfully associated with more thorough monitoring of patients or with a higher percentage of patients achieving therapeutic anticoagulation. There was moderately wide variation in therapeutic ranges among the 140 sites. CONCLUSIONS: The prevention of heparin over-anticoagulation represents an important opportunity for improving patient safety in a significant number of institutions.
LUPUS ANTICOAGULANT
The effect of lupus anticoagulant in the second-generation assay for activated protein C resistance. Ragland BD, Reed CE, Eiland BM, Tichenor PH, Hudson CL, Fritsma GA, Adler BK, Marques MB. University of Alabama at Birmingham, USA	Am J Clin Pathol 2003 Jan;119(1):66-71 Abstract quote The activated protein C resistance (APCR) assay is the test of choice to screen for factor V Leiden. We evaluated the effect of lupus anticoagulant on the baseline clotting time of the second-generation APCR assay with plasma samples from 54 patients to determine whether a falsely low APCR ratio could be predicted. We also assessed whether a modification of the assay could make it more reliable in the presence of strong lupus anticoagulants. Of 54 plasma samples, 5 yielded a false-positive APCR ratio, and all 5 had a prolonged baseline clotting time. Further dilution (1:40) of the plasma samples in factor V-deficient plasma led to correction of the APCR ratio and did not affect the sensitivity of the test for factor V Leiden. Our data support that the baseline clotting time is a good predictor of a false-positive APCR test result and should be checked before calculating the ratio. The modified APCR assay reliably identified the false-positive ratios and could be used to screen for factor V Leiden in samples with strong lupus anticoagulant.
MIXING STUDIES
A "Percent Correction" Formula for Evaluation of Mixing Studies Sheng-hsiung Chang, MD Veronica Tillema, MT(ASCP) and Doris Scherr, MT(ASCP)	Am J Clin Pathol 2002;117:152-155 Abstract quote The study examined a "percent correction" formula for evaluating mixing study results comparing a 1:1 mix with a new 4:1 mix of patient plasma with citrated normal plasma for a prolonged activated partial thromboplastin time (aPTT) and/or prothrombin time (PT). The study also examined 3 suggested definitions of correction for evaluating mixing study results for comparison. Applicability of percent correction for evaluating the aPTT 4:1 mix testing with and without incubation also was studied. Our results showed that percent correction of the aPTT or PT 4:1 mix had an overall good sensitivity and specificity for detecting anticoagulant and factor deficiency and was better than that of the aPTT or PT 1:1 mix. The 3 suggested definitions of correction all had a poor sensitivity for detecting anticoagulant. The percent correction of the aPTT 4:1 mix testing after incubation had better sensitivity and specificity that that of testing immediately. Nevertheless, these procedures were complementary to each other. The percent correction using the aPTT or PT 4:1 mix seemed to offer a simple, objective, and effective criterion for evaluating mixing study results.
PLATELET COUNTING
Platelet Counting by the Coulter LH 750, Sysmex XE 2100, and Advia 120 A Comparative Analysis Using the RBC/Platelet Ratio Reference Method Linda M. Sandhaus, MD Ebenezer S. Osei, MS Neeta N. Agrawal, MD Christine A. Dillman, MT(ASCP) Howard J. Meyerson, MD	Am J Clin Pathol 2002;118:235-241 Abstract quote We compared the accuracy and precision of the impedance platelet counts generated by the Beckman Coulter LH 750 and the Sysmex XE 2100 and the optical platelet counts produced by the Advia 120 and the Sysmex XE 2100 with flow cytometric reference platelet counts. Samples analyzed had platelet counts less than 150 × 103/µL (150 × 109/L) with a platelet flag or less than 75 × 103/µL (75 × 109/L) on the Sysmex SE 9500. The 105 samples were run sequentially through each analyzer. Anti-CD41 and anti-CD61 monoclonal antibodies were used for flow cytometric determination of the reference platelet count by the RBC/platelet ratio method. The Beckman Coulter and the Sysmex impedance platelet counts showed better correlation with the reference method than the optical platelet counts by the Advia and the Sysmex. At platelet transfusion thresholds of 10 and 20 × 103/µL (10 and 20 × 109/L), the precision of the impedance methods was somewhat better than that of the optical methods. Current methods of optical platelet counting may not be superior to impedance platelet counts for all patient populations.
POINT OF CARE TESTING
Coagulation and Transfusion Medicine / Original Article Point-of-Care Testing for Prothrombin Time, but Not Activated Partial Thromboplastin Time, Correlates With Laboratory Methods in Patients Receiving Aprotinin or epsilon-Aminocaproic Acid While Undergoing Cardiac Surgery Tak-Shun Choi, MD Philip E. Greilich, MD Chen Shi, MD James S. Wilson, MA Amy Keller, CLS and Martin H. Kroll, MD	Am J Clin Pathol 2002;117;74-78 Abstract quote Point-of-care testing (POCT) of coagulation parameters can help optimize transfusion practice in cardiac surgery. Antifibrinolytic agents may interfere with the laboratory and/or POCT coagulation assays. This randomized controlled study compared coagulation parameters obtained from a whole blood POCT coagulation device with a typical laboratory instrument in cardiac surgery patients receiving aprotinin, epsilon-aminocaproic acid, or normal saline before undergoing cardiopulmonary bypass. Aliquots of arterial blood samples from 42 patients were collected perioperatively, and their prothrombin times (PTs) and activated partial thromboplastin times (aPTTs) were measured by POCT and laboratory instrumentation. Linear regression and error analyses were used for the method comparison. For PT, the POCT device compared favorably with the laboratory method. For aPTT, the POCT device did not compare well with the laboratory method. Treatment with antifibrinolytic agents does not interfere with determination of PT.
PROTEIN S ASSAYS	Am J Clin Pathol 2005;123:778-785
REPTILASE TIME
Elevated Fibrinogen in an Acute Phase Reaction Prolongs the Reptilase Time but Typically Not the Thrombin Time Elizabeth M. Van Cott, MD Eve Y. Smith Dennis K. Galanakis, MD	Am J Clin Pathol 2002;118:263-268 Abstract quote The effects of elevated fibrinogen on thrombin and reptilase times have not been well documented. High fibrinogen levels are common (38% of specimens submitted to our coagulation laboratory). Among 102 patients in the present study, an endogenously elevated fibrinogen level was significantly associated, as follows, with prolonged reptilase times: 1 (4%) of 28 with normal fibrinogen levels, 6 (20%) of 30 with levels in the 400 to 700 mg/dL (4.0-7.0 g/L) range, 10 (34%) of 29 with levels in the 700 to 1,000 mg/dL (7.0-10.0 g/L) range, and 7 (47%) of 15 with fibrinogen levels greater than 1,000 mg/dL (10.0 g/L). This association was independent of patient age and fibrin degradation product titer. In contrast, thrombin time was not altered notably by elevated fibrinogen levels. In 4 patients studied further, the prolonged clotting times could be corrected or nearly corrected by adding calcium chloride or albumin, whereas no such corrections were demonstrable in samples from several hereditary dysfibrinogenemia control subjects. An elevated fibrinogen level is common and is associated with reptilase time prolongations. For patients with prolonged reptilase times, a fibrinogen assay is suggested before establishing a diagnosis of dysfibrinogenemia.
THROMBOPHILIA
Laboratory investigation of thrombophilia. Tripodi A, Mannucci PM. Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine, University and IRCCS Maggiore Hospital, Via Pace 9, 20122 Milan, Italy.	Clin Chem 2001 Sep;47(9):1597-606 Abstract quote Until recently, laboratory diagnosis of thrombophilia was based on investigation of the plasmatic anticoagulant pathways to detect antithrombin, protein C, and protein S deficiencies and on the search for dysfibrinogenemia and anti-phospholipid antibodies/lupus anticoagulants. More recently, laboratory investigations have been expanded to include activated protein C (APC) resistance, attributable or not to the presence of the factor V Leiden mutation; hyperprothrombinemia attributable to the presence of the prothrombin gene mutation G20210A; and hyperhomocysteinemia attributable to impairment of the relevant metabolic pathway because of enzymatic and/or vitamin deficiencies. All of the above are established congenital or acquired conditions associated with an increased risk of venous and, more rarely, arterial thrombosis. Testing is recommended for patients who have a history of venous thrombosis and should be extended to their first-degree family members. Because most of the tests are not reliable during anticoagulation, it is preferable to postpone laboratory testing until after discontinuation of treatment. Whenever possible, testing should be performed by means of functional assays. DNA analysis is required for the prothrombin gene mutation G20210A. Laboratory diagnosis for anti-phospholipid antibodies/lupus anticoagulant should be performed by a combination of tests, including phospholipid-dependent clotting assays and solid-phase anti-cardiolipin antibodies. Hyperhomocysteinemia can be diagnosed by HPLC methods or by fluorescence polarization immunoassays.
TISSUE PLASMINOGEN ACTIVATOR
Tissue plasminogen activator plasma levels as a potential diagnostic aid in acute pulmonary embolism. Flores J, Garcia-Avello A, Flores VM, Navarro JL, Canseco F, Perez-Rodriguez E. Servicio de Neumologia, Hospital Universitario Principe de Asturias, Alcala de Henares, Madrid, Spain.	Arch Pathol Lab Med 2003 Mar;127(3):310-5 Abstract quote CONTEXT: Pulmonary embolism (PE) is a potentially fatal and frequent complication of deep venous thrombosis, and the most reliable techniques for the diagnosis of PE are not universally available and have other limitations. OBJECTIVE: To determine the efficacy of 4 different fibrinolysis system parameters, namely, tissue plasminogen activator (tPA), tissue plasminogen activator inhibitor type 1 (PAI-1), plasmin-antiplasmin complexes (PAP), and D-dimer, in the diagnosis of acute PE. SETTING: A 350-bed university hospital serving an area with 280,000 inhabitants. PATIENTS: Sixty-six consecutive outpatients with clinically suspected PE. The diagnosis of PE was based on ventilation-perfusion (V/Q) lung scan in combination with clinical assessment, lower limb study, and (when required) pulmonary angiography. MAIN OUTCOME MEASURES: At the moment of clinical suspicion, a sample of venous blood was obtained to measure levels of tPA, PAI-1, PAP, and D-dimer using an enzyme-linked immunosorbent assay method. RESULTS: Twenty-seven patients (41%) were classified as PE positive (high clinical probability and V/Q lung scan [n = 12], nondiagnostic V/Q lung scan and high clinical probability [n = 1], inconclusive V/Q lung scan and positive lower limb examination for deep venous thrombosis [n = 11], and positive pulmonary angiography [n = 3]), and 39 patients (59%) were classified PE negative. The sensitivity/negative predictive value for tPA, using a cutoff of 8.5 ng/mL, and PAI-1, using a cutoff of 15 ng/mL, were 100%/100% and 100%/100%, respectively. A tPA level lower than 8.5 ng/mL occurred in 13 (19.7%; all PE negative) of 66 patients with suspected PE, and PAI-1 levels were lower than 15 ng/mL in 9 (13.6%; all PE negative) of 66 patients with suspected PE. The D-dimer, using a cutoff of 500 ng/mL, showed a sensitivity and negative predictive value of 92.6% and 87.5%, respectively. CONCLUSIONS: Our data indicate that tPA and PAI-1 levels are potentially useful in ruling out PE, although tPA seems to be the better parameter. The sensitivity levels and negative predictive values for the rapid enzyme-linked immunosorbent assay for D-dimer used in this investigation were low compared with previous studies using the same test.

 

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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Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.

Basic Principles of Disease
Learn the basic disease classifications of cancers, infections, and inflammation

Commonly Used Terms
This is a glossary of terms often found in a pathology report.

Diagnostic Process
Learn how a pathologist makes a diagnosis using a microscope

Surgical Pathology Report
Examine an actual biopsy report to understand what each section means

Special Stains
Understand the tools the pathologist utilizes to aid in the diagnosis

How Accurate is My Report?
Pathologists actively oversee every area of the laboratory to ensure your report is accurate

Got Path?
Recent teaching cases and lectures presented in conferences

• Pathologists Who Make A Difference
• Search for a Physician Specialist

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