Mark T. Cunningham

Laboratory Diagnosis of Dysfibrinogenemia

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in abnormal fibrinogen function. It can be inherited or acquired. The inherited form is associated with increased risk of bleeding, thrombosis, or both in the same patient or family. Traditionally, dysfibrinogenemia is diagnosed by abnormal tests of fibrin clot formation; the thrombin time and reptilase time are the screening tests, and the fibrinogen clotting activity-antigen ratio is the confirmatory test. The inherited form is diagnosed by demonstrating similar laboratory test abnormalities in family members, and if necessary by analysis of the fibrinogen protein or fibrinogen genes in the patient. The acquired form is diagnosed by demonstrating abnormal liver function tests and by ruling out dysfibrinogenemia in family members. This article reviews the laboratory testing of dysfibrinogenemia and presents an algorithm for sequential test selection that can be used for diagnosis.

Effect of fondaparinux on coagulation assays: Results of College of American pathologists proficiency testing

CONTEXT Fondaparinux, a factor Xa inhibitor, is approved for thromboprophylaxis after orthopedic surgery and for treatment of venous thromboembolism. It may also be efficacious, safe, and cost-effective for other patients; thus, more widespread use of fondaparinux is likely. The effect of fondaparinux on coagulation testing needs to be thoroughly examined. OBJECTIVE To report the effects of fondaparinux on coagulation tests (prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, factor VIII, thrombin time, anti-factor Xa) across diverse methodologies. DESIGN Samples with different concentrations of fondaparinux (0, 0.4, 0.8, and 2.0 microg/mL) were sent to laboratories participating in the College of American Pathologists Comprehensive Coagulation proficiency survey (N = 898). Laboratory-specific methods were used to assay coagulation parameters. RESULTS Prophylactic or therapeutic fondaparinux prolonged the prothrombin time by approximately 1 second and the activated partial thromboplastin time by 4 to 5 seconds, and reduced factor VIII from 119% to 107% and 102%, respectively. Supratherapeutic fondaparinux reduced factor VIII to 85%. The activated partial thromboplastin time was prolonged in 19%, 29%, and 52% of laboratories with prophylactic, therapeutic, and supratherapeutic fondaparinux levels, respectively. Fibrinogen, antithrombin, and thrombin time assays did not show clinically significant changes. When measuring fondaparinux concentration using an anti-factor Xa assay, the most accurate results were obtained when fondaparinux was used as the calibrator. CONCLUSIONS Fondaparinux, even in prophylactic doses, slightly prolongs the prothrombin time and activated partial thromboplastin time and can interfere with factor VIII assays, but it has no clinically relevant effect on fibrinogen, antithrombin, or thrombin time. A fondaparinux standard curve should be used for reporting fondaparinux levels using an anti-factor Xa assay.

Laboratory reporting of the international normalized ratio: Progress and problems

CONTEXT The international normalized ratio (INR) is widely used to monitor oral anticoagulation and to evaluate patients with coagulation disorders. OBJECTIVE To examine the variability of the performance and reporting of the INR and to evaluate laboratory calculation of the INR. DESIGN Between 1993 and 2003, laboratories participating in proficiency testing were surveyed. Participants provided the international sensitivity index and the mean normal prothrombin time used to calculate the INR. The INR was calculated from the data provided and compared with the INR reported to determine if the calculation was correct. RESULTS Survey data regarding the INR collected between 1993 and 2003 demonstrate an improvement in reporting, using appropriate anticoagulant, using lower international sensitivity index reagents, and matching international sensitivity index and prothrombin time method. The all-method coefficient of variation of the INR improved from 18% to 5.8%. Among 3813 laboratories studied in 2002 and 2003, 4.1% miscalculated INR. Of 29 laboratories that reported investigation of the INR miscalculation, 11 (38%) reported correcting an INR that was being reported in patient results and that this error was corrected as a result of the study. Since beginning grading of the INR calculation, miscalculation of the INR has fallen to less than 1%. CONCLUSIONS Recommendations for change in laboratory practice made by consensus conferences are implemented during the course of many years. Difficulty calculating the INR was documented, and both the calculation and the variability in the reporting of the INR showed improvement. Proficiency testing, when closely evaluated and acted on, can have a direct impact on the quality of patient care.

External quality assurance of fibrinogen assays using normal plasma: Results of the 2008 college of american pathologists proficiency testing program in coagulation

CONTEXT Proper diagnosis and therapy of fibrinogen deficiency requires high-quality fibrinogen assays. OBJECTIVE To assess the interlaboratory bias, precision, and grading of fibrinogen assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in coagulation. DESIGN Two identical vials of normal plasma were sent to more than 3500 laboratories. Participants measured fibrinogen levels using local methods. RESULTS Fifty different fibrinogen methods were evaluated. All-method bias was 8.3% (range of method-specific biases, 0.0%-27.0%) and all-method coefficient of variation was 7.7% (range of method-specific coefficients of variation, 0.7%-25.8%). After controlling for reagent/instrument type, mean fibrinogen levels were 11.6% higher for prothrombin time-based reagents compared to Clauss (P < .001), and coefficient of variation was 46% lower for mechanical endpoint instruments compared to photo-optical. Most testing events (97.4%) could be reliably graded as pass or fail using a target range of ±20% from the method mean (total pass rate, 98.8%). Total fail rate was 3.0-fold lower for mechanical instruments compared to photo-optical (0.5% versus 1.5%, P  =  .001). Nonetheless many photo-optical methods had very high precision and very low fail rates. CONCLUSIONS Fibrinogen assays showed highly variable methodology and performance characteristics. Bias, precision, and grading were affected by the type of reagent or instrument used.

Laboratory heparin resistance in burn injury complicated by venous thrombosis

Anticoagulation with heparin is required in the management of the burn patient if their clinical course is complicated by venous thrombosis. Heparin therapy is commonly monitored by the activated partial thromboplastin time (APTT) but this assay can be unreliable in patients with acute inflammation because of an increase in plasma factor VIII levels that result in an underestimation of the heparin concentration. We report an example of heparin resistance that occurred in a patient who developed venous thrombosis following extensive second-degree burns. Heparin doses in excess of 60,000 units per day were required to produce a significant elevation in the APTT. The plasma factor VIII level was found to be markedly elevated to 455% and the plasma heparin concentration as determined by the anti-factor Xa assay was disproportionately elevated in relation to the APTT. Physicians treating patients with burn injury complicated by venous thrombosis should be aware of the potential development of factor VIII-related heparin resistance when large amounts of heparin are required to obtain a satisfactory elevation in the APTT. Measurement of the plasma heparin concentration will avoid excessive heparin administration and the serious bleeding which can result.

External Quality Assurance of Antithrombin, Protein C, and Protein S Assays Results of the College of American Pathologists Proficiency Testing Program in Thrombophilia

CONTEXT Hereditary and acquired deficiencies of antithrombin (AT), protein C (PC), and protein S (PS) are risk factors for venous thromboembolism. Proper diagnosis requires high-quality assays for these proteins. OBJECTIVE To determine the accuracy and interlaboratory precision of AT, PC, and PS assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in thrombophilia and to grade the performance of laboratories. DESIGN Standardized normal plasma with assigned analyte values was sent in 2 separate challenges to participating laboratories. Participants measured AT, PC, and PS levels using local methods. RESULTS When compared with the assigned values for the international standard, the order of assay accuracy from highest to lowest was AT activity, PC antigen, AT antigen, total PS antigen, PC activity, PS activity, and free PS antigen (range of assay bias, 2.6%-8.8%). The order of assay precision from highest to lowest was PC activity, AT activity, AT antigen, total PS antigen, PS activity, free PS antigen, and PC antigen (range of assay coefficient of variation, 6.1%-20.0%). Most testing events (87.8%) could be graded as pass or fail using a target range of ±3 standard deviations from the method-specific mean. The pass rate was 98.2% for all AT, PC, and PS testing events combined. CONCLUSIONS Accuracy and precision were higher for AT assays and lower for PC and PS assays. It was feasible to grade individual laboratory performance.

The clonality of CD3+ CD10+ T cells in angioimmunoblastic T cell lymphoma, B cell lymphoma, and reactive lymphoid hyperplasia.

T cells coexpressing CD3 and CD10 are a characteristic feature of angioimmunoblastic T-cell lymphoma (AITL) [1]. However, they are not unique to AITL, as these cells are also present in B cell lymphoma and reactive lymphoid hyperplasia [2]. To determine the significance of CD3+ CD10+ T cells, we used flow cytometry with cell sorting and molecular biology techniques for T cell gene rearrangement to study T cells from patients with AITL, B cell lymphoma, and reactive lymph node hyperplasia. We found that CD3+ CD10+ T cells in B cell lymphoma and reactive lymphoid hyperplasia were polyclonal. In early stage of AITL, they were oligoclonal, and became monoclonal as AITL progressed. These findings illustrate the differences between early and late lymphoma and could be important for the diagnosis of AITL.

Evidence of a Phospholipid Binding Species within Human Fibrinogen Preparations

Fibrinogen has been reported to interact with phospholipid; however, the properties of this binding interaction have not been characterized. Purified preparations of human fibrinogen bound to small unilamellar vesicles containing phosphatidylserine (PS) as measured by light scattering and radioisotope filtration. Binding to 100% PS was saturable (apparent Kd=5 microM, Bmax=1.9 g protein/g lipid), reversible, and involved a minor subfraction of the fibrinogen preparation (3-6% of total protein). Fibrinogen interacted minimally with phosphatidylinositol, and not at all with pure phosphatidylcholine (PC) or PC vesicles containing 5% glycosphingolipid (lactosylceramide, ganglioside GM3, ganglioside GD3). Binding efficiency decreased as the PS content of vesicles was diluted with PC. Calcium chloride (2 mM) enhanced protein binding to PS, which was reversed by EDTA. Fibrin clot formation almost quantitatively precipitated the PS binding activity. PS, but not PC, increased the final turbidity of fibrin clots. Computerized sequence analysis of fibrinogen revealed three candidate acidic phospholipid binding motifs located at position 143-210 in the alpha chain, and positions 59-77 and 101-139 in the beta chain. Further study of the PS binding activity of fibrinogen may lead to new insights about fibrinogen function.

Abnormal Wnt signaling and stem cell activation in reactive lymphoid tissue and low-grade marginal zone lymphoma

The variable natural history of mucosa-associated lymphoid tissue (MALT) lymphoma poses a challenge in predicting clinical outcome. Since Wnt signaling, as indicated by nuclear localization of β-catenin, is believed to be key in stem cell activation and stem cell self-renewal, we explored the possibility that it might have a predictive value in marginal zone lymphoma. We chose to analyze pβ-catenin-S552 because its nuclear localization by immunohistochemistry appears to coincide with Wnt signaling-initiated tumorigenesis in intestinal and hematopoietic tissues. Wnt signaling and activation was studied in 22 tissue samples of extranodal marginal zone lymphoma, atypical lymphoid hyperplasia, reactive lymphoid hyperplasia, and normal lymphoid tissue to determine whether Wnt signaling could help distinguish MALT lymphoma from benign lesions. Compared to normal or reactive lymphoid tissue, we found increased nuclear expression of localized pβ-catenin-S552 in atypical lymphoid hyperplasia and extranodal marginal zone lymphoma. We show that the anti-pβ-catenin-S552 antibody may be useful in diagnosing and monitoring the progression of or response to therapy of MALT lymphoma.

FATAL DURAL SINUS THROMBOSIS ASSOCIATED WITH HETEROZYGOUS FACTOR V LEIDEN AND A SHORT ACTIVATED PARTIAL THROMBOPLASTIN TIME

Inherited thrombophilia is a risk factor for dural sinus thrombosis (DST). To our knowledge, this is the first description with autopsy findings of a patient with DST associated with heterozygous factor V Leiden and a short activated partial thromboplastin time (aPTT). A 51-year-old woman presented with a 3-day history of headache, nausea, right-sided weakness, and focal motor seizure; she died 3 days after admission. At autopsy, a gross examination showed hemorrhage of bilateral parietal lobes and left primary motor cortex, uncal and tonsillar herniation, and pulmonary embolus of the right upper lobe. A microscopic examination of the brain showed an organizing thrombus in the superior sagittal sinus, diffuse cerebral edema, and extensive venous congestion. Laboratory studies showed heterozygous factor V Leiden by polymerase chain reaction and a very short aPTT of 17 seconds (reference range, 22-30 seconds). The combination of a heterozygous factor V Leiden mutation and a short aPTT may have contributed to the fatal DST in this patient.