CD44 as a Potential Screening Marker for Preliminary Differentiation Between Congenital Dyserythropoietic Anemia Type II and Hereditary Spherocytosis.

BACKGROUND: Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method. METHODS: Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed. RESULTS: The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells. CONCLUSIONS: A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins.

INTRODUCTION: Hereditary spherocytosis (HS) and hereditary pyropoikilocytosis (HPP, severe form of hereditary elliptocytosis) are unrelated red cell disorders caused by defects in distinct regions of the red cell cytoskeleton. The high predictive value of the eosin-5-maleimide (EMA)-binding test for the diagnosis of HS is because of its interaction with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47, which are reduced on HS red cells. Our study was undertaken to determine why EMA-labelled HPP red cells were previously found to give much lower fluorescence readings than HS. METHODS: Flow cytometry was used to determine the relative amounts of monoclonal antibodies bound to red cells from normal adults, HS and HPP groups. Confocal microscopy was used to visualise the overall staining pattern of the red cells with selected antibodies. RESULTS: In flow cytometry, HPP red cells gave lower antibody binding to the four EMA-reactive membrane proteins than HS red cells and bound less antibody to glycophorins A and C, and CD59. Confocal images of Rh protein and band 3 immunostaining revealed a greater number of HPP red cells having partial or no fluorescence than in HS and normal controls. CONCLUSION: Lesser amounts of EMA-reactive membrane proteins were detected in HPP than HS red cells, thus confirming their lower fluorescence readings in the EMA-binding test. The concomitant reduction in glycophorins A and C, and CD59 in HPP could have caused cellular contraction, resulting in poikilocytosis.

Assessment of the clinical utility of the rim and comet-tail signs in differentiating ureteral stones from phleboliths.

OBJECTIVE: This study was designed to assess interobserver variability in identifying the rim and comet-tail signs and to determine the clinical utility of these signs in determining whether or not the calcifications with which they are associated represent ureteral calculi. MATERIALS AND METHODS: Two radiologists and a radiology resident, unaware of the final diagnosis, reviewed preselected helical CT images from renal stone examinations in patients with 65 indeterminate pelvic calcifications. Assessment of calcifications for rim or comet-tail signs was performed independently of an assessment for the following five secondary signs of urinary tract obstruction: caliectasis, pelviectasis, ureterectasis, perinephric stranding, and renal enlargement. Agreement in identifying rim and comet-tail signs was assessed by obtaining kappa statistics. The utility the of rim or comet-tail signs in determining whether ureterolithiasis was present in patients in whom perinephric stranding and ureterectasis were present or absent was determined. The frequency with which one or more of each of the five assessed secondary signs was identified ipsilateral to a calcification having rim or comet-tail signs was also tabulated. RESULTS: Kappa values for interobserver agreement ranged from 0.49 to 0.73. In only one patient was a rim sign detected in the absence of ureterectasis and perinephric stranding. Reviewers identified at least three of the five assessed secondary signs ipsilateral to calcifications showing a rim sign in all but one patient (by each radiologist) and four patients (by the resident). When three or more secondary signs of obstruction were seen ipsilateral to a calcification having a comet-tail sign, in all but one instance, this was because the calcification was a ureteral calculus or because there was a separate ipsilateral ureteral calculus. CONCLUSION: In many instances, observers did not agree about whether the rim and comet-tail signs were present. The rim sign was observed in the absence of any secondary signs of urinary tract obstruction in only one (1.5%) of the 65 patients in our series (95% confidence interval, 0-5.3%). The comet-tail sign, when accompanied by secondary signs of obstruction, should indicate that an ipsilateral ureteral stone is present and not the reverse.

Adverse effects of increased body weight on quantitative measures of mammographic image quality.

OBJECTIVE: The purpose of our study was to show that compressed breast thickness on mammograms in overweight and obese women exceeds the thickness in normal-weight women and that increased thickness results in image degradation. SUBJECTS AND METHODS: Three hundred consecutive routine mammograms were reviewed. Patients were categorized according to body mass index. Compression thickness, compressive force, kilovoltage, and milliampere-seconds were recorded. Geometric unsharpness and contrast degradation were calculated for each body mass index category. RESULTS: Body mass index categories were lean (3%), normal (36%), overweight (36%), and obese (25%). Body mass index was directly correlated with compressed thickness. In the mediolateral oblique view, the mean thickness of the obese category exceeded normal thickness by 18 mm (p < 0.01), corresponding to a 32% increase in geometric unsharpness. Mean obese thickness exceeded lean thickness by 33 mm (p < 0.01), corresponding to a 79% increase in unsharpness. Similar trends were observed for the craniocaudal view. In the mediolateral oblique projection, there was an increase of 1.0 kVp (p < 0.01) for obese compared with normal and 1.7 kVp (p < 0.01) between lean and obese, corresponding, respectively, to a 16% and a 25% decrease in image contrast because of scatter and kilovoltage changes. Milliampere-seconds increased by 47% on the mediolateral oblique images in the obese category compared with normal body mass index. CONCLUSION: An increased body mass index was associated with greater compressed breast thickness, resulting in increased geometric unsharpness, decreased image contrast, and greater potential for motion unsharpness.

Progressive esophageal leiomyomatosis with respiratory compromise.

Leiomyomatosis is a rare neoplastic condition of the pediatric esophagus. Presenting symptoms usually overlap with more common esophageal disorders, namely, gastroesophageal reflux. A patient is presented in whom leiomyomatosis progressed to the point of causing cachexia and respiratory compromise.

Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry.

The use of flow cytometry for quantifying fetomaternal haemorrhage is increasing, and has been shown to be more accurate than the Kleihauer-Betke test for evaluating larger bleeds of over 4 mL in volume. Red cells are stained with fluorescently labelled monoclonal anti-D. Cells for analysis are normally gated manually on the basis of forward and side scatter. We investigated whether the use of an antiglycophorin A monoclonal antibody conjugate (red cell specific) in a dual labelling technique would improve the gating of RBC and FMH quantification. Mixes of adult rr and cord R1r RBC were prepared to simulate 1, 0.5, 0.25, 0.12 and 0.06% fetal bleeds. Phycoerythrin-conjugated BRIC 256 (mouse monoclonal antiglycophorin A) was used to label all RBC, and FITC-BRAD-3 monoclonal anti-D was used to determine the proportion of D-positive cells. Results from the dual labelling experiments were compared to those from single labelling of the same mixtures with FITC-BRAD-3 alone, using gated and ungated data. The results showed that single labelling with manual gating gave falsely low FMH estimates. We conclude that use of a fluorescently labelled antiglycophorin A antibody improves the accuracy of the FMH measurement by flow cytometry, as manual subjective gating of RBC excludes a higher proportion of fetal than of adult RBC.

Detection of weak D and D(VI) red cells in D-negative mixtures by flow cytometry: implications for feto-maternal haemorrhage quantification and D typing policies for newborns.

Quantitation of feto-maternal haemorrhage (FMH) by flow cytometry (FC) has been shown to be more accurate than the Kleihauer-Bekte test. Fetal cells will be predominately of R1r or R2r phenotype, with antigen site numbers per cell (SPC) of between 9900 and 16000. If the fetus is of weak D or partial D(VI) phenotype, fewer SPC will be present. Red cells from 20 adult weak D samples were mixed with rr red cells to give 1% mixes. Mixtures were stained and analysed by FC, using two different monoclonal reagents. The SPC of each sample was measured using SOL-ELSA with Scatchard plot analysis. 18 samples could not be distinguished and had <1000 SPC. Two samples that could be distinguished had 1350 and 3000 SPC. Red cells from seven samples of D(VI) were also analysed. None of these samples could be distinguished: SPC were all <1000. Although one of the reagents used reacts with D(VI) cells, quantitation of a D(VI) FMH would not be possible due to low SPC. The ability of fetal red cells with low Rh D SPC to cause immunization is questionable; failure to measure FMH in these cases is unlikely to cause clinical problems, as long as suitably sensitive serological reagents and techniques are used to type all weak D and D variant babies as Rh D positive, and thus ensure that the mother is given the appropriate dose of anti-D.

Limitations of student evaluations of curriculum.

RATIONALE AND OBJECTIVES: Medical student surveys are used extensively in the development and modification of curriculum. The purpose of this study was to look at medical student surveys of a radiology lecture series, evaluating the accuracy of student perceptions of learning and factors affecting them. MATERIALS AND METHODS: After a "Case of the Week" lecture series, 156 3rd-year medical students returned a survey evaluating the experience with 10 questions on a four-point scale (1 = disagree, 4 = agree very much) and took a clinical competency assessment (CCA) examination with a radiology substation. Survey responses were compared with actual examination performance, analyzed for how overall learning was characterized in specific educational objectives, and evaluated for factors affecting perceived learning. RESULTS: The mean response for perceived CCA examination preparedness was 1.83. The mean radiology station test score was 90.43%. Correlations between student perception of learning and the scoring of focused learning objectives ranged from 0.33 to 0.48 (P < .01). Students responding 1 to items assessing perceived lecture organization, stimulation to read, and interest in the field of radiology had mean scores for perception of overall learning of 2.09-2.44 and mean scores for recommendation of course continuation of 1.68-2.46. Students responding 4 had means of 3.25-3.81 and 3.06-4.0, respectively. CONCLUSION: Student perceptions of the value of curriculum were inaccurate compared with external measures of performance, and students poorly related their general impressions to specific learning objectives. Perceived lecture organization, stimulation to read, and interest in radiology as a specialty affected perceived overall learning and perceived value of the lecture series.

Novel C-2 substituted carbapenem derivatives. Part IV. Synthesis and biological activity of five membered heteroaromatic derivatives.

The synthesis, antibacterial activity, and stability to human dehydropeptidase-1 (DHP-1) of a novel series of (5R,6S)-6-[(1R)-1-hydroxyethyl]-2-heterocyclylcarbapen-2-em-3-carb oxylates are described. Of the compounds investigated 1,5-disubstituted pyrazol-3-yl and 3-substituted isoxazol-5-yl derivatives have the best combination of antibacterial activity and stability to DHP-1. They are particularly active against community-acquired respiratory tract pathogens and have stabilities to DHP-1 superior to that of meropenem.

The ecology of race and socioeconomic distress: infant and working-age mortality in Chicago.

We examine the effects of education, unemployment, and racial segregation on age-, sex-, and race-specific mortality rates in racially defined Chicago community areas from 1989 to 1991. Community socioeconomic factors account for large observed areal variations in infant and working-age mortality, but especially working-age mortality for the black population. For black men, the mortality consequences of living in economically distressed communities are quite severe. Segregation effects on mortality are more modest and largely operate through neighborhood socioeconomic conditions, although some direct effects of segregation on mortality for blacks are apparent.

Novel C-2 substituted carbapenem derivatives. Part I. Synthesis and biological activity of non-aromatic heterocyclic derivatives.

A new series of carbapenems, having a saturated or partially unsaturated heterocycle at C-2, has been synthesised. The in vitro antibacterial activity of these compounds and their stability to human dehydropeptidase-1 (DHP-1) are described. The stereochemistry of the C-2 side-chain and the presence of a double bond in the heterocycle were shown to have significant effects on the stabilities of the compounds to DHP-1.

Structure-activity relationships within a series of C(7)-substitutedoxyiminocephalosporins containing the C(3)-methylaminopyridiniumthiomethyl substituent. Synthesis and biological properties of BRL 57342 and some close analogues.

(6R,7R)-7-[2-(2-Amino-4-thiazolyl)-2-[(Z)-[(S)-carboxy(3,4- dihydroxyphenyl)methyl]oxyimino]acetamido]-3-(1-methylaminopyri dinium-4-thiomethyl)ceph-3-em-4-carboxylate sodium salt (BRL 57342, 1f) combines excellent in vitro antibacterial potency against Gram-positive and Gram-negative bacteria, including P. aeruginosa and Acinetobacter spp., with excellent stability to extended spectrum beta-lactamases. This potency is reflected in in vivo efficacy studies.

Synthesis and biological activity of 3-(N-substituted pyridinium-4-thiomethyl)-7 alpha-formamido cephalosporins.

The synthesis and antibacterial activity of a series of 3-(1-substituted pyridinium-4-thiomethyl)-7 alpha-formamido cephalosporins is described. All the derivatives showed good potency and stability to bacterial beta-lactamases. The antibacterial efficacy seen with the N-alkyl pyridinium substituents was enhanced by the introduction of a catecholic side chain at C-7 and by preparation of N-(substituted amino)pyridinium derivatives.

Synthesis and biological properties of some 3-[(N-substituted-amino)pyridinium-4-thiomethyl]-7-[2-(2-amino-thiazol- 4-yl)-2-(Z)-(methoxyimino)acetamido]ceph-3-em-4-carboxylates.

The synthesis and antibacterial activity of a series of beta-lactamase stable, broad spectrum 7-[2-(2-amino-thiazol-4-yl)-2-(Z)-(methoxyimino)acetamido]-cephalo sporins, characterised by a C-3-[N-(substituted-amino)pyridinium-4-thiomethyl] group, is described. Gram-positive and Gram-negative bacteria including extended spectrum beta-lactamase-producing strains were most susceptible to the N-amino- and N-methylamino derivatives (3a) and (3b); with the exception of Pseudomonas aeruginosa, (3b) was more active in vitro and in vivo than cefpirome or ceftazidime.

Quantitation of monoclonal antibodies by ELISA. The use of purified mouse IgG and mouse IgM monoclonal antibodies as standards in a quantitative ELISA measuring monoclonal antibodies produced by cell culture.

Murine monoclonal antibodies (MAbs) of IgG1, IgG2a and IgG2b subclasses and IgM class in hybridoma culture supernatants were quantified using a sensitive, reliable, optimized indirect double antibody sandwich ELISA. In the ELISA, the MAb in the culture supernatants was sandwiched between affinity isolated heavy chain specific polyclonal antibodies used for capture and detection. Quantitation was achieved by comparison with a standard curve produced by a purified MAb of the same class, subclass or ideally the same clone as the MAb to be quantified. These quantitative results were compared with those obtained using purified IgG and IgM polyclonal serum samples as standards and those obtained by total protein estimation using measurement at OD280nm. The IgG subclass MAbs used as standards were purified using protein G and the IgM class MAb was purified by ion exchange followed by gel filtration chromatography. Bovine IgG contamination of the MAb supernatants and the purified MAbs was also measured by a double antibody sandwich ELISA.

Analysis of the structure and activity of A and A,B immunoglobulin A monoclonal antibodies.

A study of the serologic activity and molecular structures of three spleen-derived mouse IgA monoclonal human blood group-specific supernatants was undertaken; this was part of an evaluation of these monoclonals as blood typing reagents. The monoclonal antibodies were eluted through a precalibrated size-exclusion column, and fractions were analyzed by immunoblotting, heavy and light chain-specific enzyme-linked immunosorbent assay, and liquid- and solid-phase serologic tests. Results indicated that one of the supernatants (anti-A specificity) contained tetrameric and monomeric forms of IgA, while the other two (anti-A,B specificity) contained three higher polymeric forms (1000-4000 kDa) and one dimeric form. The tetrameric and polymeric forms showed red cell agglutinating activity, whereas the dimeric and monomeric forms did not. All forms contained heavy and light chains. The monomeric anti-A showed specific binding to appropriate red cells in a solid-phase assay, but the dimeric anti-A,B fractions did not. Purified fractions stored at 4 degrees C did not show any equilibration toward other forms, which indicated that the molecules are stable once secreted. The use of such antibodies as blood grouping reagents requires careful monitoring to ensure that high proportions of nonagglutinating molecular weight forms are not produced, as they could compromise the performance of the reagent by binding to red cell antigen in competition with the agglutinating forms.

Chemical crosslinking of proteins of the influenza virion. 1. Interrelationships.

Purified influenza virus (A/FPV/Rostock/34; H7N1) was reacted with one of three chemical crosslinking reagents [dimethylsuberimidate (DMS), tartryl diazide (TDA) and formaldehyde] under conditions designed to give a ladder of crosslinked polypeptides (putative homo- and heteropolymers) when analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions. The different virion polypeptides were identified by Western blotting with monospecific antisera against HA1, HA2, NP, and M1. When reacted with any crosslinker NP preferentially formed 2mer and 4mer homopolymers while M1 formed 2mers, 4mers, 6mers, and 8mers. 2mers and 3mers of HA1 were detected after crosslinking with TDA and DMS but homopolymers of HA2 could not be identified with certainty due to comigrating M1. One heteropolymer was clearly identified as 1NP:1M1 (with DMS and TDA) and others, as expected, as components of the haemagglutinin spike 1HA1:1HA2, 2HA1:2HA2, and 3HA1:3HA2. Formaldehyde gave rise only to HA1:HA2 polymers. The presence of other heteropolymers containing NP in conjunction with HA2 and HA1 seemed likely. Whenever HA2 ran with an Mr of about 50k it comigrated with M1 suggesting it may have formed (with DMS or TDA) a 1HA2:1M1 heterodimer. However it is possible that this band consisted of HA2 homodimers comigrating with M1 homodimers. Patterns of crosslinking with DMS and TDA were similar although not identical, but those obtained with formaldehyde were markedly different. All patterns were highly reproducible.

Chemical cross-linking of proteins of the influenza virion. 2. Acid-induced irreversible conformational changes in HA1 and HA2.

Purified influenza virus (A/FPV/Rostock/34;H7N1) was exposed briefly to pH 5 before returning to an alkaline pH. Virus was then reacted with one of three chemical cross-linking reagents [dimethyl suberimidate (DMS), tartryl diazide (TDA), or formaldehyde which span 11, 6, and 2A, respectively]. Cross-linked polypeptides were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and identified with monospecific antisera against HA1, HA2, NP and M1. Acidification resulted in changes in the cross-linking patterns for both HA1 and HA2 which could be detected with all three reagents. Most notable were the data with formaldehyde: under alkaline conditions cross-linking gave only HA1:HA2 heteropolymers but after brief acidification none of these were formed and in their place was a novel HA1 homodimer, an HA2 homotrimer and an HA2 of Mr 50k cross-linked to form a homodimer with another HA2 or to a heterodimer with M1. Although cross-linking by formaldehyde was much more affected by acidification of the virus than cross-linking by DMS or TDA, over half the polymers cross-linked by DMS were no longer formed after acidification. The patterns of cross-linking of NP and M1 were unchanged by low pH treatment.