Vanlila K. Swami

Intimal hyperplasia producing thrombus organization in an experimental venous thrombosis model

PURPOSE A venous thrombosis animal model demonstrated similarities between intimal hyperplasia and thrombus organization. This has prompted the evaluation of a hypothesis that intimal hyperplasia may be the mechanism for thrombus organization in veins with normal pressure. METHODS Thrombi were produced in surgically exposed jugular veins of anesthetized, 18 to 20 kg pigs. Thrombosis was induced by a combination of devascularization, electric injury produced by a low amperage, direct current, and permanent partial ligation (50% diameter reduction). Vein segments were harvested at 0, 1, 2, 7, 14, and 60 days and histologically examined for fibrin, red blood cells, platelets, smooth muscle cells, endothelial cells, elastic fibers, and collagen deposits. RESULTS Forty vein segments in 20 pigs were evaluated. Luminal thrombi with thickened walls developed in all specimens. All luminal thrombi demonstrated partial spontaneous thrombolysis over the period of observation. Intimal thickening consisting primarily of smooth muscle cells by day 2 was apparent and progressed until about 2 weeks, when collagen deposits became prominent within the neointima. The neointima frequently comprised half the cross-sectional area of the veins. Endothelial cells were present in the intima as single cells or as lining for clefts formed within the thickened intima. CONCLUSIONS Smooth muscle cell proliferation with collagen deposition characteristic of intimal hyperplasia seemed to be the mechanism of thrombus organization in the experimental thrombosis model used in this study in which extensive stimulation was used to produce thrombosis.

Age determination of experimental venous thrombi by ultrasonic tissue characterization

PURPOSE The ability of ultrasonic tissue characterization based on radiofrequency signal processing to detect compositional differences in thrombi of varying ages was evaluated in vivo. METHODS Thrombi were produced in 49 jugular veins of 26 anesthetized 18 to 20 kg pigs by partial ligation and application of direct electric current. Thrombi were imaged 30 minutes after formation and 1, 7, and 14 days later with a color Doppler ultrasound scanner that identified the thrombi, and acquired radio frequency data for ultrasonic tissue characterization analysis. Ultrasonic tissue characterization used two parameters from the normalized power spectrum, slope, and intercept, which are related to scatterer size, scatterer concentration, and acoustic-impedance differences between scatterers and surrounding medium. Previous in vitro studies demonstrated that lower slope and higher intercept values correlated with greater cellularity and more-dense fibrin mesh. Histologic examination was performed for each time period. The values of slope and intercept for each timed observation were compared by a multilinear discriminant analysis. RESULTS There were no statistical differences between day 0 and day 1. Statistically-significant differences in ultrasonic tissue characterization parameters were seen between all other time intervals with p values < 0.01. Older thrombi tended to demonstrate higher slope and lower intercept values. These ultrasonic tissue characterization changes correlated with a red cell and fibrin-mesh density reduction, which was confirmed by histologic findings and was indicative of partial spontaneous thrombolysis. The degree of spontaneous thrombolysis provides an estimate of the age of thrombi. CONCLUSION Ultrasonic tissue characterization is capable of distinguishing age differences in thrombi in an animal model and has the potential for noninvasive application in clinical diagnosis.

Specific immunoadsorption of IgG antibody in a patient with chronic lymphocytic leukemia and autoimmune hemolytic anemia: A new form of therapy for the acute critical stage☆

Progressive and severe autoimmune hemolytic anemia developed in a patient with chronic lymphocytic leukemia (CLL) despite treatment with chlorambucil, high doses of corticosteroids and attempts to transfuse packed red blood cells. Splenectomy was not performed because of severe coronary artery disease. Direct antiglobulin tests revealed a warm red blood cell autoantibody of IgG-type with anti-e specificity. The patient was treated by extracorporeal immunoadsorption of plasma IgG using a cell separator and protein A as the immunoadsorbent. The patient responded by an increase in the hemoglobin levels and platelet counts after two treatments. Specificity of the procedure was shown by a decrease in the serum IgG and by the demonstration of the same reactivity to ficin-treated reagent red blood cell panel of the eluate from the protein A. Antibody titers of the patients red blood cell eluate decreased from 1:128 to 1:64 and eventually the anti-e specificity was lost. This is a report of a novel approach to treatment of the acute phase of an autoimmune hemolytic anemia.

Ultrasonic Tissue Characterization of Blood Clots

Ultrasonic tissue characterization based on an analysis of the power spectrum of backscattered signals obtained with ultrasound was used to distinguish morphologic components of blood clots. The three morphologic features for which discrimination was attempted were loose fibrin, red-cell, and dense fibrin clots. The UTC was able to distinguish the morphologic blood components tested. This in vitro work was based on the analysis of parameters related to ultrasound-tissue interaction and on inferences related to the physical properties of scatterer properties (scatterer size, scatterer concentration, and ratio of scatterer to medium acoustic impedances). The ability to distinguish these blood-clot components suggests that UTC may be able to distinguish red from white thrombi and to assess the structures and changes within thrombi associated with the age of the thrombus, their mechanical properties, and treatment monitoring.

Assessing Breast Cancer Margins Ex Vivo Using Aqueous Quantum-Dot-Molecular Probes

Positive margins have been a critical issue that hinders the success of breast- conserving surgery. The incidence of positive margins is estimated to range from 20% to as high as 60%. Currently, there is no effective intraoperative method for margin assessment. It would be desirable if there is a rapid and reliable breast cancer margin assessment tool in the operating room so that further surgery can be continued if necessary to reduce re-excision rate. In this study, we seek to develop a sensitive and specific molecular probe to help surgeons assess if the surgical margin is clean. The molecular probe consists of the unique aqueous quantum dots developed in our laboratory conjugated with antibodies specific to breast cancer markers such as Tn-antigen. Excised tumors from tumor-bearing nude mice were used to demonstrate the method. AQD-Tn mAb probe proved to be sensitive and specific to identify cancer area quantitatively without being affected by the heterogeneity of the tissue. The integrity of the surgical specimen was not affected by the AQD treatment. Furthermore, AQD-Tn mAb method could determine margin status within 30 minutes of tumor excision, indicating its potential as an accurate intraoperative margin assessment method.

Cellularity and fibrin mesh properties as a basis for ultrasonic tissue characterization of blood clots and thrombi.

This in vitro study was designed to evaluate the ability of ultrasonic tissue characterization (UTC) based on power spectrum analysis of backscattered radio-frequency echo signals to distinguish two prominent variables of thrombi: cellularity (primarily red cell content) and fibrin-mesh density. Six types of clots simulating thrombus components were prepared by varying red-cell and platelet concentrations and shear forces during clotting. Data were acquired with a linear-array transducer, digitized, and analyzed in terms of slope and intercept parameters obtained from normalized power spectra of radio-frequency echo signals. Increased cellularity and fibrin-mesh density both produced lower slope and higher intercept values, which permitted statistically significant discrimination of cellularity and mesh density in the six types of clots analyzed. Shearing forces and (to a lesser degree) platelet concentrations increased fibrin-mesh density. This study suggests that UTC based upon the power spectrum of echo signals may be used to detect and follow compositional differences that have clinical relevance in the diagnosis and follow-up of thrombi.

Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots

In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative.

Ultrasonic tissue characterization of experimental venous intimal hyperplasia.

Ultrasonic tissue characterization (UTC) employing slope and Y-intercept parameters from the normalized power spectrum of backscattered echoes was employed in vivo to study compositional changes in the walls of pig jugular veins in which thrombi were experimentally induced. Light microscopy revealed these changes to be intimal hyperplasia with an early predominance of smooth muscle cells and a later mixture of smooth muscle cells and collagen deposits. UTC distinguished intimal hyperplasia from previously reported data from luminal thrombosis UTC. Furthermore, UTC was able to discriminate between early (predominantly smooth muscle cells) and older (smooth muscle cells plus collagen deposits) intimal hyperplasia. The study suggests that intimal hyperplasia in the experimental model used may be organized thrombus and that UTC may be able to follow both the development of wall changes as well as luminal changes occurring in venous thrombosis.

Aberrant Blood Grouping Results in a Patient with Splenomegaly and Thrombocytopenia

Principal Laboratory Findings [T1] and [T2] Questions: 1. What is (are) this patient’s most striking laboratory result(s)? 2. How do you explain this patient’s most striking laboratory result(s)? 3. What is this patient’s blood group and Rh type? 4. What other tests can be done to confirm this patient’s blood group and the presence of hypogammaglobulinemia? 5. What are other possible causes of ABO discrepancies?

Spurious Thrombocytosis in a Burn Patient

Questions: 1. What is (are) this patient’s most striking laboratory result(s)? 2. How do you explain this patient’s most striking laboratory result(s)? 3. What does the instrument-generated flag “LURI” indicate? 4. What additional test(s) should be done to confirm your answer to question #3? 5. How do you explain this patient’s markedly elevated platelet count? 6. What other conditions are associated with spurious thrombocytosis? 7. What conditions are associated with spurious thrombocytopenia?