Assessment of coronary flow reserve with transthoracic Doppler echocardiography: comparison with intracoronary Doppler method

To evaluate the feasibility and usefulness of transthoracic Doppler echocardiography (TTDE) as a non-invasive method in recording distal anterior descending (LAD) coronary flow velocity, we compared coronary flow reserve (CFR) measured by TTDE with measurements by intracoronary Doppler wire (ICDW). Twenty-one patients without LAD stenosis were studied. ICDW performed at baseline and after intracoronary injection of 18 microg adenosine. TTDE was performed at baseline and after intravenous adenosine (140 microg/kgmin for 2 min). Adequate Doppler recordings of coronary flow velocities during systole were obtained in 14 of 21 study patients (67%) and during diastole in 17 (81%) patients. Baseline and hyperemic peak diastolic flow velocities measured by TTDE were significantly smaller than those obtained by ICDW (p<0.05). However, diminishing trends of diastolic and systolic velocity ratio after hyperemia were similarly observed in both methods. CFR obtained by TTDE (3.0+/-0.5), was higher than the value calculated by ICDW (2.5+/-0.4). There were significant correlations between the values obtained by the two methods (r=0.72, p<0.01). It is concluded that TTDE is a feasible method in measuring coronary flow velocity and appears to be a promising non-invasive method in evaluating CFR.     

T lymphocyte activation and restenosis after percutaneous transluminal coronary angioplasty.

We investigated the relation between the activation of T lymphocytes and the occurrence of restenosis after percutaneous transluminal coronary angioplasty (PTCA) in 10 stable angina patients. Recent studies have suggested that PTCA causes an inflammatory response, which may affect restenosis after angioplasty. Soluble interleukin-2 receptor (sIL-2R) is a useful marker to evaluate the activation of T lymphocytes. sIL-2R was measured before and 2 h after successful PTCA, and 3-month follow-up coronary angiography was done to observe restenosis. Four of 10 patients showed restenosis. The restenosis group of 4 patients had a higher level of sIL-2R after PTCA than the no-restenosis group of 6 patients (495 vs. 274 U/ml, p < 0.01). This study suggests that sIL-2R may offer prognostic information after elective PTCA and identify a subgroup of patients at high risk for clinical restenosis in a few months.     

Mechanisms of restenosis after coronary intervention: difference between plain old balloon angioplasty and stenting.

BACKGROUND: Restenosis after coronary intervention remains an unsolved and important clinical problem. We histologically examined the mechanism of restenosis after both balloon injury and stenting. METHODS: Coronary arteries of swine were subjected to balloon injury and stenting. Next, just after stenting or at 7, 14, or 28 days, the animals were sacrificed for the evaluation by morphometric analysis, histological observation, and immunostaining. RESULTS: The neointimal area peaked at 14 days in the balloon injury group (BG) and increased linearly up to 28 days in the stent group (SG). At 28 days, the total vascular area in the BG was reduced to 78% of the control values. In the SG, the total vascular area remained enlarged. According to the phenotypic analysis, the vascular smooth muscle cells (VSMCs) in the neointimal area at 28 days were the contractile type in the BG and the synthetic type in the SG. Proliferating cell nuclear antigen (PCNA) and macrophage-positive cells were not observed in neointima in the BG at 28 days, whereas they were observed around the stent struts in the SG. In addition, numerous inflammatory cells, such as neutrophils and eosinophils, were also present in the SG. CONCLUSIONS: Restenosis after balloon injury consisted of arterial remodeling and neointimal hyperplasia, whereas that after stenting consisted mostly of neointimal hyperplasia. The neointimal area in the SG lasted longer than that in the BG. Continuous inflammation may be an important factor in the restenosis of stenting.     

Image analysis can be used to detect spatial changes in the histopathology of pancreatic tumours

Pancreatic cancer is frequently associated with intense growth of fibrous tissue at the periphery of tumours, but the histopathological quantification of this stromal reaction has not yet been used as a prognostic factor because of the difficulty of obtaining quantitative measures using manual methods. Manual histological grading is a poor indicator of outcome in this type of cancer and there is a clinical need to establish a more sensitive indicator. Recent pancreatic tumour biology research has focused upon the stromal reaction and there is an indication that its histopathological quantification may lead to a new prognostic indicator. Histological samples from 21 cases of pancreatic carcinoma were stained using the sirius red, light-green method. Multiple images from the centre and periphery of each tumour were automatically segmented using colour cluster analysis to subdivide each image into representative colours. These were classified manually as stroma, cell cytoplasm or lumen in order to measure the area of each component in each image. Measured areas were analysed to determine whether the technique could detect spatial differences in the area of each tissue component over all samples, and within individual samples. Over all 21 cases, the area of stromal tissue at the periphery of the tumours exceeded that at the centre by an average of 10.0 percentage points (P < 0.001). Within individual tumours, the algorithm was able to detect significantly more stroma (P < 0.05) at the periphery than the centre in 11 cases, whilst none of the remaining cases had significantly more stromal tissue at the centre than the periphery. The results demonstrate that semi-automated analysis can be used to detect spatial differences in the area of fibrous tissue in routinely stained sections of pancreatic cancer.     

Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay.

Epidemiologic application of the human in vivo hypoxanthine-guanine phosphoribosyltransferase (hprt) mutation assay requires screening of mutant colonies to differentiate independent from clonal origin. Previously, sibship was defined by Southern blot analysis of T cell receptor gene rearrangements. We report here a more expedient method to determine these rearrangements utilizing the polymerase chain reaction (PCR) and a DNA single-strand conformation polymorphism technique. The results are consistent with those obtained by Southern blotting in that sibship can be defined easily. A major advantage is that cells may be taken directly from the microtiter plate, eliminating the necessity to expand the clones and isolate genomic DNA. Cell lines which have not undergone receptor gene rearrangements cannot serve as PCR templates and do not interfere with this analysis. Furthermore, background from the large number of nonmutant lymphocytes present in the well does not hinder the analysis of the T cell receptor pattern of a mutant. This technique facilitates rapid screening of a large number of clones in a shorter time than Southern blotting, and is useful for the study of in vivo mutation and the clonal expansion of mutants in populations of T cells.     

Endoluminal beta-radiation therapy for the prevention of coronary restenosis after balloon angioplasty. The Dose-Finding Study Group

BACKGROUND: Beta radiation is effective in reducing vascular neointimal proliferation in animals after injury caused by balloon angioplasty. However, the lowest dose that can prevent restenosis after coronary angioplasty has yet to be determined. METHODS: After successful balloon angioplasty of a previously untreated coronary stenosis, 181 patients were randomly assigned to receive 9, 12, 15, or 18 Gy of radiation delivered by a centered yttrium-90 source. Adjunctive stenting was required in 28 percent of the patients. The primary end point was the minimal luminal diameter six months after treatment, as a function of the delivered dose of radiation. RESULTS: At the time of follow-up coronary angiography, the mean minimal luminal diameter was 1.67 mm in the 9-Gy group, 1.76 mm in the 12-Gy group, 1.83 mm in the 15-Gy group, and 1.97 mm in the 18-Gy group (P=0.06 for the comparison of 9 Gy with 18 Gy), resulting in restenosis rates of 29 percent, 21 percent, 16 percent, and 15 percent, respectively (P=0.14 for the comparison of 9 Gy with 18 Gy). At that time, 86 percent of the patients had had no serious cardiac events. In 130 patients treated with balloon angioplasty alone, restenosis rates were 28 percent, 17 percent, 16 percent, and 4 percent, respectively (P=0.02 for the comparison of 9 Gy with 18 Gy). Among these patients, there was a dose-dependent enlargement of the lumen in 28 percent, 50 percent, 45 percent, and 74 percent of patients, respectively (P<0.001 for the comparison of 9 Gy with 18 Gy). The rate of repeated revascularization was 18 percent with 9 Gy and 6 percent with 18 Gy (P=0.26). CONCLUSIONS: Intracoronary beta radiation therapy produces a significant dose-dependent decrease in the rate of restenosis after angioplasty. An 18-Gy dose not only prevents the renarrowing of the lumen typically observed after successful balloon angioplasty, but actually induces luminal enlargement.     

Multiple coronary artery-left ventricular microfistulae in a patient with apical hypertrophic cardiomyopathy: a demonstration by transthoracic color Doppler echocardiography

Among the congenital coronary artery fistulae, multiple coronary artery microfistulae arising from the left and right coronary artery and emptying into the left ventricle are very rare and little is known of their anatomic and clinical features, especially in apical hypertrophic cardiomyopathy. A 67-year- old woman was referred for the evaluation of chest pain at exertion, and shortness of breath. Electrocardiographic and echocardiographic findings were typical of apical hypertrophic cardiomyopathy. Coronary arteriography showed normal epicardial coronary arteries, but multiple coronary artery-left ventricular microfistulae arising from the left and right coronary arteries. Transthoracic color Doppler echocardiography, using a high frequency transducer with a low Nyquist limit, demonstrated multiple coronary artery-left ventricular microfistulae just beneath the apical impulse window.     

Coronary flow reserve: measurement with transthoracic Doppler echocardiography is reproducible and comparable with positron emission tomography.

Detection of early vascular changes indicated by lowered coronary flow reserve (CFR) would allow early treatment and prevention of atherosclerosis. The purpose of this study was to test whether it is possible to reproducibly measure CFR with transthoracic Doppler echocardiography (TTE) in healthy volunteers. We measured CFR using dipyridamole infusion in ten healthy male volunteers with two methods: TTE and positron emission tomography (PET) with oxygen-15-labelled water (group A). However, CFR was assessed twice with TTE in eight healthy male volunteers (group B) to study the reproducibility of this method. We compared CFRs obtained using TTE flow measurements in the left anterior descending coronary artery (LAD) and PET flow measurements in the corresponding myocardial area. Coronary flow in LAD could be measured in all subjects using TTE. By TTE, an average CFR based on peak diastolic flow velocity (PDV) was 2.72 +/- 1.16, mean diastolic flow velocity (MDV) 2.56 +/- 1.06 and velocity time integral (VTI) 1.87 +/- 0.49. The results were reproducible in two repeated TTE studies (coefficient of variation in MDV 6.1 +/- 4.3%, n=8). By PET, CFR was 2.52 +/- 0.84. CFR assessed by TTE correlated closely with that measured by PET (MDV r=0.942, P<0.001; PDV r=0.912, P<0.002 and VTI r=0.888, P<0.006) and intraclass correlation was 0.929 (MDV) and tolerance limits for differences of CFRs was -0.78 to 0.72. We show that CFR measured by TTE has an excellent correlation with CFR measured by PET. We also found that TTE measurements of CFR were highly reproducible.     

Fluorescence in situ hybridization for the Y-chromosome can be used to detect cells of recipient origin in allografted hearts following cardiac transplantation.

The purpose of this study was to determine if fluorescence in situ hybridization for the Y-chromosome can be used to detect cells of recipient origin in allografted hearts following cardiac transplantation. Formalin-fixed, paraffin-embedded tissue sections of coronary arteries from two hearts surgically explanted from heart transplant recipients undergoing retransplantation because of accelerated arteriosclerosis were examined by fluorescence in situ hybridization for the presence of cells containing the Y-chromosome using a biotinylated Y-chromosome cocktail probe. In both cases, the recipients were male and the original donor hearts were obtained from female donors. Hybridization was detected in cells morphologically recognizable as infiltrating lymphocytes, macrophages, and mast cells, establishing that these cells in the donor hearts were of recipient origin. In contrast, hybridization was not detected in cardiac myocytes, in vascular smooth muscle cells, or in the majority (>95%) of endothelial cells, suggesting that these cells were of donor origin. Although hybridization was detected in rare flattened cells lining vascular lumina, these cells did not stain for factor VIII, suggesting that they were, in fact, flattened inflammatory cells and not endothelial cells. These results demonstrate that, when the recipient and donor are of the opposite sex, fluorescence in situ hybridization for the Y-chromosome can be used to detect graft chimerism in transplanted hearts.     

Measures of leucine aminopeptidase can be used to anticipate UV-induced age-related damage to lens proteins: ascorbate can delay this damage

This paper focuses on damage to soluble lens proteins during ultraviolet (UV) light exposure and its prevention by ascorbate (Vitamin C). Using 2.3 X 10(-3) W/cm2 UV A and 0.4 X 10(-4) W/cm2 UV B, aminopeptidase inactivation in lens supernatants is significant after 60 min. Protein aggregation and decreases in tryptophan levels, phenomena associated with UV-induced and cataract-related damage, are observed only after longer (6 h) UV exposure. Thus, it would appear that measurements of aminopeptidase activity can be used to anticipate damage to lens structural proteins. Ascorbate (15 mM) added to soluble lens proteins prior to photoirradiation can prevent some of these changes. The data presented suggest plausible relationships between impaired proteolysis and cataract formation.     

Frozen stored murine hybridoma cells can be used to determine the virulence of Listeria monocytogenes.

Murine hybridoma cells, designated Ped-2E9, when stored up to 60 days at -196 degrees C or up to 48 days at -80 degrees C, gave results equivalent to those for freshly grown murine hybridoma cells in an in vitro pathogenicity assay of Listeria species. Thus, laboratories do not need to have their own tissue culture facilities to maintain the hybridoma cells for the assay described.     

Proposal for clinical trials using anti-inflammatory drugs in the therapy of angina pectoris, myocardial infarction and coronary restenosis after angioplasty and bypass grafting.

The importance of inflammatory phenomena in atherosclerosis is now appreciated. Here, a clinical trial to be conducted using anti-inflammatory drugs (sulfasalazine, griseofulvin and colchicine) in angina pectoris, myocardial infarction and coronary restenosis after angioplasty and bypass grafting is proposed. Patients who have both atherosclerosis and a disease responsive to anti-inflammatory drugs (ulcerative colitis or Crohn's disease, dermatomycosis, necrotizing vasculitis, Behcet's disease, gout or other colchicine-sensitive diseases), are desirable targets of the present proposal.     

Immunohistochemistry can be used to subtype acute myeloid leukemia in routinely processed bone marrow biopsy specimens. Comparison with flow cytometry

Flow cytometry (FC) is the preferred method of immunophenotyping acute myeloid leukemia (AML). However, there are situations in which FC is unavailable and in which immunohistologic staining of bone marrow biopsy specimens can be used to provide immunophenotypic information. To evaluate immunohistologic staining and to confirm its value, we selected 80 newly diagnosed cases of AML that were classified according to French-American-British (FAB) criteria and confirmed by flow cytometric analysis for this study. Paraffin-embedded bone marrow specimens were stained using a panel of antibodies that included CD34 (QBEND10), antimyeloperoxidase (anti-MPO), antihemoglobin, factor VIII-related antigen, and 3 epitopes of CD68 (HAM56, KP1, and PG-M1). Our findings suggest that with the use of the paraffin-reactive antibodies CD34 (QBEND10), MPO, CD68 (PG-M1), antihemoglobin, and factor VIII-related antigen, immunohistochemistry can be used to subclassify AML. Comparison of immunohistochemical results with FC immunophenotyping suggests that there is significant concordance in the results for markers that can be used with both techniques, indicating that the sensitivity and specificity of both methods is comparable (P > .53 in all cases).     

Validation of a single survey that can be used for case identification and assessment of asthma control: the Breathmobile Program.

BACKGROUND: Underdiagnosis of asthma and underrecognition of disease severity in lower socioeconomic populations continue to be significant health care concerns despite national efforts to better educate health care providers. OBJECTIVE: To validate a 1-page survey as a point-in-time tool identifying uncontrolled vs controlled asthma and moderate-to-severe disease activity in an urban, lower-socioeconomic pediatric population. METHODS: A previously validated survey (the Breathmobile Case Identification Survey) was evaluated as a point-in-time tool for identifying children with poorly controlled disease. Clinical validation was achieved in children (n = 1,826) presenting to a school-based asthma program for either an initial (n = 666) or a follow-up (n = 1,170) visit. Responses were compared with a comprehensive evaluation by a physician specialist as the gold standard. Response patterns were used to construct multimodel tiered scoring algorithms for baseline and follow-up visits that identify children with uncontrolled asthma, and children are likely to have moderate-to-severe disease activity at that time. RESULTS: Surveys scored using the developed algorithms identified children with uncontrolled asthma (sensitivity: baseline, 77.0%; follow-up, 71.6%; specificity: baseline, 72.7%; follow-up, 71.5%) and detected moderate-to-severe disease activity (sensitivity: baseline, 69.2%; follow-up, 77.4%; specificity: baseline, 70.2%; follow-up, 70.3%). CONCLUSIONS: The Breathmobile Case Identification Survey can be used in lower-socioeconomic, urban populations as a point-in-time tool for identifying children with uncontrolled vs controlled asthma and moderate-to-severe disease activity.     

Hepatocyte paraffin 1: a monoclonal antibody that reacts with hepatocytes and can be used for differential diagnosis of hepatic tumors.

Hepatocyte paraffin 1 is a monoclonal antibody that has been developed specifically to react with hepatocytes in routine formalin-fixed and paraffin-embedded surgical pathology tissues. It results in a distinct, granular cytoplasmic staining of hepatocytes but fails to react with bile ducts and nonparenchymal liver cells. The antibody decorates a majority of hepatocellular carcinomas, including fibrolamellar variants. It fails to react with a wide variety of other adult malignancies, with the exception of focal staining in a few gastrointestinal malignancies, including a subpopulation of gastric carcinomas.     

Antibodies to distinct epitopes on the CD40 molecule co-operate in stimulation and can be used for the detection of soluble CD40.

The B-cell surface protein, CD40, belongs to the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family and plays a crucial role in T cell-dependent B-cell activation. Ligation of this receptor with antibodies or its recently defined ligand, gp39, generates an intracellular signal that, when combined with triggering of surface immunoglobulin or the interleukin-4 (IL-4) receptor, induces a variety of stimulatory effects in B cells. In this study we provide further evidence for the importance of receptor cross-linking in generating this signal and we also report on the presence of a soluble form of CD40. A new CD40 monoclonal antibody (mAb), 17:40, was found to synergize with other CD40 antibodies (mAb89 and S2C6) in inducing proliferation as well as IgE synthesis in IL-4-treated tonsillar B cells. However, both this mAb and mAb89 failed to co-operate with a soluble construct of the CD40 ligand, whereas such co-operation was seen with the S2C6 antibody. Cross-inhibition experiments showed that the 17:40 mAb recognized an epitope that was clearly distinct from that seen by S2C6 and mAb89. Although directed to separate epitopes, both 17:40 and mAb89 completely blocked binding of gp39 to its receptor, while the S2C6 mAb only partially interfered with this binding. The findings suggest a close relationship between the degree of receptor clustering and the strength of the delivered signal. With the access to antibodies recognizing distinct structures on CD40 we also established a sandwich enzyme-linked immunosorbent assay for quantitative determinations of the antigen. With this assay we could demonstrate the presence of a soluble form of CD40 (sCD40) in culture supernatants. The fact that sCD40 also retained its ligand-binding capacity indicates that it may have an important regulatory role and modulate the T cell-dependent stimulation via CD40. Both the finding of soluble receptors and the need for receptor clustering are features that CD40 share with other members of the TNF/NGF receptor family.     

The changing landscape of HLA typing: Understanding how and when HLA typing data can be used with confidence from bench to bedside

Human leukocyte antigen (HLA) genes are extraordinary for their extreme diversity and widespread impact on human health and disease. More than 30,000 HLA alleles have been officially named and more alleles continue to be discovered at a rapid pace. HLA typing systems which have been developed to detect HLA diversity have advanced rapidly and are revolutionizing our understanding of HLA’s clinical importance. However, continuous improvements in knowledge and technology have created challenges for clinicians and scientists. This review explains how differences in HLA typing systems can impact the HLA types that are assigned. The consequences of differences in laboratory testing methods and reference databases are described. The challenges of using HLA types that are not equivalent are illustrated. A fundamental understanding of the continual expansion of our understanding of HLA diversity and limitations in some of the typing data is essential for using typing data appropriately in clinical and research settings.     

Technical report: results of immunological tests on faecal extracts are likely to be extremely misleading.

Clinical investigation of gut immunity is difficult because of the need to study intestinal tissues or secretions directly. Others have reported that immunoglobulins, antibodies and cytokines can be detected in saline extracts of faeces. We have assessed the validity of this approach by measuring immunoglobulins, albumin, alpha 1-antitrypsin and isotype-specific antibodies in matched samples of faeces and whole gut lavage fluid. Results were compared as estimated output per day, and by using haemoglobin as a common reference substance. Samples were obtained from 10 patients with active inflammatory bowel disease and 10 with other benign GI diseases. For immunoglobulins, albumin and antibodies, the amount detected in faeces varied from < 0.01% to 35.5% (based on estimated daily output) and < 0.01% to 18.5% (based on haemoglobin) of the amount known to be produced in the gut from results of assays on whole gut lavage fluid (WGLF); there were significantly higher rates of recovery in faecal specimens from patients with active gut inflammation than from other patients. Detection rates and titres of specific antibody in faeces were even lower than those for immunoreactive IgA. These data indicate that immunological tests on saline extracts of faeces do not represent the true status of the gut humoral immune system, and such studies should be strongly discouraged.     

One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation

PurposeTo evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions.MethodsAn interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)–based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients.ResultsIn the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests.ConclusionThe analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.     

Presence of copper- and zinc-containing superoxide dismutase in commensal Haemophilus haemolyticus isolates can be used as a marker to discriminate them from nontypeable H. influenzae isolates

Respiratory isolates of Haemophilus haemolyticus are regularly misclassified as nontypeable (NT) Haemophilus influenzae due to an aberrant hemolytic reaction on blood agar, with implications for treatment. The presence of sodC or its cognate protein, copper-zinc superoxide dismutase, can distinguish respiratory isolates of H. haemolyticus from NT H. influenzae with 100% accuracy.