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Blood Specimens (blood + specimen)
Kinds of Blood Specimens Selected AbstractsIn vivo mutation assay based on the endogenous Pig-a locusENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2008Steven M. Bryce Abstract The product of the X-chromosome's Pig-a gene acts in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, and is thereby essential for attaching certain proteins to the cell surface. The experiments described herein were designed to evaluate whether lack of GPI-anchored proteins could form the basis of an in vivo mutation assay. Specifically, we used a CD59-negative cell surface phenotype to denote Pig-a mutation. Besides anti-CD59-PE, two other fluorescent reagents were used: thiazole orange to differentiate mature erythrocytes, reticulocytes (RETs), and leukocytes; and anti-CD61 to resolve platelets. These experiments were performed with Sprague Dawley rats, and focused on two cell populations, total erythrocytes and RETs. The ability of the analytical method to enumerate CD59-negative erythrocytes was initially assessed with reconstruction experiments whereby mutant-mimicking cells were added to control bloods. Subsequently, female rats were treated on three occasions with the model mutagens ENU (100 mg/kg/day) or DMBA (40 mg/kg/day). Blood specimens were harvested at various intervals, as late as 6 weeks post-exposure. Considering all week 4,6 data, we found that CD59-negative cells ranged from 239 to 855 × 10,6 and 82 to 405 × 10,6 for ENU and DMBA, respectively. These values were consistently greater than those observed for negative control rats (18 ± 19 × 10,6). The elevated frequencies observed for the genotoxicant-exposed animals were usually higher for RETs compared to total erythrocytes. These data support the hypothesis that an efficient in vivo mutation assay can be developed around flow cytometric enumeration of erythrocytes and/or RETs that exhibit aberrant GPI-anchored protein expression. Environ. Mol. Mutagen., 2008. © 2008 Wiley-Liss, Inc. [source] Human metapneumovirus and respiratory syncytial virus infections in older children with cystic fibrosisPEDIATRIC PULMONOLOGY, Issue 1 2007Daniel F. Garcia MD Abstract Background: Human metapneumovirus (hMPV) has been isolated from children with acute respiratory infection worldwide. Its epidemiology remains to be defined in children with cystic fibrosis (CF). We describe the epidemiology and clinical impact of hMPV in CF children and compared it to respiratory syncytial virus (RSV). Methods: CF children ages 7,18 years were studied prospectively during the 1998,1999 RSV season. Nasopharyngeal specimens were collected during acute respiratory illnesses and tested for respiratory viruses. Blood specimens were drawn early, mid, and end of the RSV season, and tested for serological evidence of hMPV and RSV infections. Rates of lower respiratory tract illnesses (LRTI) and hospitalizations for pulmonary exacerbations were compared during the time intervals they developed serological evidence of infection to their non-infection intervals. Results: Six of 44 CF children had a virus positive respiratory illness in 56 LTRI events and 18 hospitalizations. Serological evidence of hMPV and RSV infections occurred in 16 and 20 CF children, respectively; 8 had infections with both viruses. A greater proportion of CF children had ,1 LRTI during their infection intervals compared to their non-infection intervals (13/25 vs. 5/25; P,=,0.03). A trend for higher rates of LRTI was observed in the infection intervals compared to non-infection intervals (9.5,±,11.0 vs. 4.2,±,9.9 per 1,000 child-days; P,=,0.06), and it was significantly greater with a more conservative estimate (one event per child per interval; 7.4,±,7.7 vs. 2.6,±,5.4 per 1,000 child-days; P,,0.01). No differences in hospitalizations rates were detected. Conclusion: The infection rates and clinical impact observed for hMPV were comparable to that for RSV in CF children 7,18 years of age. Pediatr Pulmonol. 2007; 42:66,74. © 2006 Wiley-Liss, Inc. [source] Changes in serum leptin concentration after corticosteroid treatment in preterm infantsACTA PAEDIATRICA, Issue 6 2002PC Ng The aim of this study was to investigate the effect of postnatal systemic dexamethasone on serum leptin, insulin and hormones of the hypothalamic-pituitary-adrenal (HPA) axis in preterm, very low birthweight (VLBW) infants. Nineteen VLBW infants who received a 3 wk dose tapering course of dexamethasone for treatment of bronchopulmonary dysplasia were prospectively enrolled. Blood for hormone assays was collected immediately before the start of the dexamethasone course (Td-pre), 3 wk after commencement of the drug (Td-end) and 2 wk after dexamethasone treatment had been stopped (Td-post). In addition, 28 VLBW infants who participated in a concurrent longitudinal leptin study within the same period but did not receive corticosteroid had their serum leptin and insulin concentrations serially monitored. Blood specimens for the latter group of infants were obtained at 2 (Twk,2), 5 (Twk,5) and 7 (Twk,7) wk of postnatal age. Serum leptin and insulin at Td,end were significantly increased, whereas plasma ACTH and serum cortisol were significantly suppressed compared with the pretreatment (Td,pre) levels in the corticosteroid group (p > 0.0001 for leptin and insulin; p > 0.05 and p > 0.001 for ACTH and cortisol, respectively). In contrast, serum leptin and insulin at weeks 5 (Twk,5) and 7 (Twk,7) did not differ significantly from their respective levels at week 2 (Twk,2) in the non-treatment group. Conclusion: The administration of systemic corticosteroid resulted in significant increases in serum leptin and insulin, but marked suppression of hormones of the HPA axis. The effect of dexamethasone on the "adipoinsular" and HPA axes was transient and reversible. The adipoinsular axis in preterm infants is likely to be functional and active at an early stage of human development, and leptin may regulate energy balance in VLBW infants in the early postnatal period. Corticosteroids may, through the adipoinsular axis or its associated pathways, mediate in the regulation of body weight in preterm neonates. [source] Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasmaLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 3 2006Esa-Matti Eino Lilius Abstract Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16,1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC50) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release. Copyright © 2006 John Wiley & Sons, Ltd. [source] In vitro, high-resolution 1H and 31P NMR based analysis of the lipid components in the tissue, serum, and CSF of the patients with primary brain tumors: one possible diagnostic viewNMR IN BIOMEDICINE, Issue 2 2010Niraj Kumar Srivastava Abstract In vitro, high-resolution 1H and 31P NMR based qualitative and quantitative analyses of the lipid components of the tissue, serum, and CSF of patients with primary brain tumors were performed. Proton NMR spectra of the lipid extract of serum (blood specimen collected before the surgical procedure) and surgically discarded tissue showed that the total cholesterol (T.CHOL) and choline containing phospholipids (PL) were significantly higher in quantity in medulloblastoma and glioblastoma multiforme as compared to normal subjects. Serum lipid extracts of grade II/ III gliomas showed a higher quantity of PL than normal subjects. Cholestrol esters (CHOLest) were detectable in the tissue lipid extract of the patients with tumors and absent in normal tissue. There was a reduction in the quantity of CHOLest in the serum lipid extract of the tumor patients as compared to normal subjects. Ratio of PL to T.CHOL in serum lipid extract showed a significant difference between different grades of tumors versus normal subjects, while, a significant difference was observed only in medulloblastoma versus normal subjects in tissue lipid extract. Ratio of CHOL to CHOLest distinguishes the different grades of tumors versus normal subjects as well as between different grades of tumors (except medulloblastoma versus glioblastoma). The ratio of the Ph (total phospholipids except phosphatidylcholine) to PC (phosphatidylcholine) in 31P NMR based study showed a significant difference in all grades of tumors (except medulloblastoma) in normal subjects in tissue lipid extract as well as between different grades of tumors. Medulloblastoma could be differentiated from glioblastoma as well as from normal subjects in serum lipid extract by the ratio of the Ph to PC. Proton NMR spectra of the lipid extract of CSF showed that the CHOL, CHOLest, and PL were present in the patients with tumors, although these were absent in the patients with meningitis, motor neuron disease, and mitochondrial myopathies as well as in normal subjects. PL and T.CHOL provided discrimination between different grades of tumors (except glioblastoma versus medulloblastoma) in the lipid extract of the CSF. This study suggests the role of lipid estimation in CSF and serum as a complementary diagnostic tool for the evaluation of brain tumors preoperatively. NMR-based lipid estimation of post-surgical tumor tissue may also contribute to differentiating the tumor types. Copyright © 2009 John Wiley & Sons, Ltd. [source] Utility of CD26 in flow cytometric immunophenotyping of T-cell lymphomas in tissue and body fluid specimens,CYTOMETRY, Issue 6 2008Diane M. Pierson Abstract Background CD26 is expressed by most CD4+ T cells in normal peripheral blood specimens. Neoplastic T cells are frequently CD26, in mycosis fungoides/Sezary syndrome involving the peripheral blood. However, CD26 expression by reactive and neoplastic T cells in solid tissues and body fluids has not been fully characterized by flow cytometry (FC). Methods Solid tissue and body fluid specimens were assayed for CD26 expression using four-color FC immunophenotyping, by qualitative assessment of population clusters, and by quantitation with comparison with isotype controls. Benign T cells were studied in reactive tissues and in the background of other malignancies. Results Many T-cell lymphomas were dim or negative for CD26, whereas a few were brightly positive. In the majority of T-cell lymphomas, CD26 expression could potentially help identify aberrant population clusters. T cells in reactive tissue specimens and tumor-infiltrating T cells were commonly dim to negative for CD26. Conclusions Both T-cell lymphomas and reactive T cells in tissue and body fluid specimens often show low levels of CD26 expression. Therefore, quantitative methods may not reliably distinguish benign from neoplastic T cells in these specimens. However, CD26, in combination with other T-cell markers, can be helpful for identifying aberrant population clusters in T-cell lymphomas. © 2008 Clinical Cytometry Society [source] An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemiaCYTOMETRY, Issue 4 2006T. Vincent Shankey Abstract Background: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. Methods: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4,17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. Results: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. Conclusions: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory. © 2006 International Society for Analytical Cytology [source] Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemiaCYTOMETRY, Issue 4 2006Rachel Sheridan Abstract Background: The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. Methods: We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19+ B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t -test. Results: A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. Conclusions: These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. © 2006 International Society for Analytical Cytology [source] Impact of the international program for quality assessment and standardization for immunological measures relevant to HIV/AIDS: QASICYTOMETRY, Issue 2 2002Francis Mandy Abstract Measurements of CD4 T-cell levels are essential for the assessment of human immunodeficiency virus (HIV) disease course, clinical staging, epidemiological studies, and decisions regarding prophylactic therapies against opportunistic infection. Until now, only in the industrialized countries was T-cell subset monitoring considered a practical option to assess disease progression. The Quality Assessment and Standardization for Immunological Measures Relevant to HIV/AIDS (QASI) program was established in 1997 to meet performance assessment for immunophenotyping laboratories in countries where such service is not available. The QASI program is provided at no cost to any laboratory in a resource-poor setting that wishes to participate. This report describes the beneficial impact of participation in the QASI program. Carefully selected commercial stabilized whole blood preparations were sent regularly to participating laboratories. Participants reported the T-cell subset values they obtained by flow cytometry. Once the aggregate mean values for the T-cell subsets were established for the shipment, a comprehensive and confidential report was sent to each laboratory. The results from five consecutive shipments were analyzed. The coefficient of variation decreased from 7.2% to 4.7% and from 14.2% to 8.8% for percent and absolute CD4 T-cell counts, respectively. With the implementation of the QASI program using commercial stabilized whole blood specimens, it is possible to reduce interlaboratory error. This study illustrates that a quality assessment program can improve the overall performance of laboratories. Reducing interlaboratory variation can enhance significantly the effectiveness of multicenter HIV vaccine or drug trial evaluation. Cytometry (Clin. Cytometry) 50:111,116, 2002. © 2002 Wiley-Liss, Inc. [source] Do ,9 -tetrahydrocannabinol concentrations indicate recent use in chronic cannabis users?ADDICTION, Issue 12 2009Erin L. Karschner ABSTRACT Aims To quantify blood ,9 -tetrahydrocannabinol (THC) concentrations in chronic cannabis users over 7 days of continuous monitored abstinence. Participants Twenty-five frequent, long-term cannabis users resided on a secure clinical research unit at the US National Institute on Drug Abuse under continuous medical surveillance to prevent cannabis self-administration. Measurements Whole blood cannabinoid concentrations were determined by two-dimensional gas chromatography-mass spectrometry. Findings Nine chronic users (36%) had no measurable THC during 7 days of cannabis abstinence; 16 had at least one positive THC ,0.25 ng/ml, but not necessarily on the first day. On day 7, 6 full days after entering the unit, six participants still displayed detectable THC concentrations [mean ± standard deviation (SD), 0.3 ± 0.7 ng/ml] and all 25 had measurable carboxy-metabolite (6.2 ± 8.8 ng/ml). The highest observed THC concentrations on admission (day 1) and day 7 were 7.0 and 3.0 ng/ml, respectively. Interestingly, five participants, all female, had THC-positive whole blood specimens over all 7 days. Body mass index did not correlate with time until the last THC-positive specimen (n = 16; r = ,0.2; P = 0.445). Conclusions Substantial whole blood THC concentrations persist multiple days after drug discontinuation in heavy chronic cannabis users. It is currently unknown whether neurocognitive impairment occurs with low blood THC concentrations, and whether return to normal performance, as documented previously following extended cannabis abstinence, is accompanied by the removal of residual THC in brain. These findings also may impact on the implementation of per se limits in driving under the influence of drugs legislation. [source] Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension,application to the variants of mannose-binding lectin gene,HUMAN MUTATION, Issue 3 2006Panayotis G. Zerefos Abstract Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.,619G>C and c.,290G>C (promoter region), and one SNP at position c.,66C>T of the 5, untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5, end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3,sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data. Hum Mutat 27(3), 279,285, 2006. © 2006 Wiley-Liss, Inc. [source] Disseminated cutaneous Fusarium moniliforme infections in a leukemic childINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 5 2007Ching-Chi Chi MD A 5-year-old boy had a 10-month remission of acute lymphocytic leukemia (ALL) after chemotherapy. Re-induction chemotherapy was performed for relapse of ALL. Thereafter, he suffered from an episode of neutropenic fever with pneumonia. One week following control of the condition with antibiotics, a 1 × 1-cm, red, painful nodule appeared on the left thigh, which was initially suspected to be Pseudomonas infection. Parenteral ceftazidime and amikacin were administered, but persistent high fever, mild cough, and a few painful erythematous papulonodules on the face and lower extremities appeared several days later (Fig. 1). These lesions increased insidiously in diameter up to 2,5 cm with central necrosis. Hemogram showed neutropenia with a shift to the left [white blood cell (WBC) count, 2.1 × 109/L; neutrophil count, 0.21 × 109/L]. A skin biopsy showed heavy growth of hyaline branching septate hyphae in the deep dermis and subcutis, together with fat necrosis (Fig. 2). Invasion of molds into vessels and sweat glands was also seen. A culture from a lesion yielded Fusarium moniliforme, but no fungi were isolated from blood specimens. Only mild infiltrations on bilateral lower lung fields were detected by chest roentgenography. The skin lesions gradually healed and the fever subsided 2 weeks after the initiation of therapy with amphotericin B 30 mg and itraconazole 200 mg daily. Figure 1. A few painful erythematous papulonodules appeared on the face and lower extremities Figure 2. Skin biopsy showed heavy growth of hyaline branching septate hyphae in the deep dermis and subcutis along with fat necrosis (hematoxylin and eosin, ×400) Meanwhile, relapse of leukemia was detected by hemogram showing atypical leukocytosis (WBC count of 24,400 × 109/L, with blast cells representing 78%). A course of chemotherapy with cytarabine, mitoxantrone, and VP-16 was prescribed, subsequently resulting in neutropenia (WBC count, < 0.1 × 109/L; neutrophil count, 0/L) and spiking fever. Although the aforementioned antifungal therapy was continued, the centers of the originally healed lesions turned dusky red, swollen, necrotic, and ulcerative. There were more than 10 such ecthymiform lesions. After administration for 22 days, itraconazole was discontinued because of no appreciable effects. Granulocyte colony-stimulating factor (G-CSF) salvage was used, and the neutropenia gradually subsided 20 days later. In addition, the ecthymiform lesions gradually resolved. Amphotericin B was discontinued 1 week following neutrophil recovery. The patient died of Acinetobacter baumannii and Stenotrophomonas maltophilia sepsis 8 months later. [source] Development and clinical application of nucleated red blood cell counting and staging on the automated haematology analyser XE-2100TMINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 1 2003F.-S. Wang Summary We initially developed a new flow cytometric (FCM) reference method for the enumeration and staging of nucleated red blood cells (NRBC) in 1997 [Wang et al., 1998 (XIth International Symposium on Technological Innovations in Laboratory Haematology, Banff, Canada, 1998); Tsuji et al., 1999 (Cytometry, 1999)]. The method used CD45 antibody and propidium iodide staining to separate NRBCs from other cells. Accuracy and precision were enhanced because larger numbers of cells were counted than was possible with the manual method. We also developed a method for automated NRBC counting on a haematology analyser, the XE-2100 (Wang, 1988). NRBC were separated from other cells using a special lysing buffer and a fluorescent dye. The XE-2100 was found to detect peripheral and cord blood NRBC accurately and precisely when compared with cell morphology or FCM control methods. The FCM NRBC staging method was established through the identification of different NRBC populations following the novel staining and lysing method. To evaluate the method further, we sorted samples containing NRBCs using a FACSort and investigated NRBC staging on the Sysmex XE-2100TM based on the cell sorting results. Data were analysed using special software (ida). First, we used the data in various parameter combinations. We then established gates to classify the NRBC populations. Finally, we analysed blood specimens from patients with different types of diseases to explore possible clinical applications. [source] Rapid screening assay of congenital adrenal hyperplasia by measuring 17,-hydroxyprogesterone with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry from dried blood spotsJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2002Chien-Chen Lai Abstract A rapid, simple, and specific method was developed for the diagnosis of congenital adrenal hyperplasia (CAH) from dried blood spots on newborn screening cards based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). The usefulness of 17,-hydroxyprogesterone (17OH-P) determination on dried filter-paper blood samples from patients with CAH caused by 21-hydroxylase deficiency was evaluated. The LC/MS/MS detection of 17OH-P was rapid, <4 min. The intra- and interday accuracy and precision of the method were <7%. Our procedure maintained good linearities (R2 > 0.992) and recovery rate (>83%). We used this new method to directly determine the 17OH-P levels in dried blood specimens from abnormal children of various ages, with a detection limit of 20 ng/ml (,240 pg), to avoid the time-consuming derivatization steps required by the gas-chromatography/mass spectrometry (GC/MS) method. Four dried filter-paper blood samples of CAH patients (three girls and one boy, 1,14 years old) were all quantified in an LC/MS/MS study and revealed high 17OH-P levels (>90 ng/ml). After treatment, all of the elevated 17OH-P levels either decreased or disappeared. Compared with CAH patients, 17OH-P was nearly undetectable (<20 ng/ml) in the normal infants by LC/MS/MS. This LC/MS/MS assay is not only useful for both diagnosis and monitoring of treatment of CAH in all other age groups, it also can be used as a screening test for CAH infants. In this study, we provided the first data on 17OH-P in dried blood specimens affected with CAH using HPLC/ESI-MS/MS. J. Clin. Lab. Anal. 16:20,25, 2002. © 2002 Wiley-Liss, Inc. [source] Analysis of mixed infections by multiple genotypes of human cytomegalovirus in immunocompromised patientsJOURNAL OF MEDICAL VIROLOGY, Issue 5 2009P. Sowmya Abstract Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised patients. The present study was carried out to determine the frequency of occurrence of multiple genotypes of HCMV in immunocompromised patients, to determine if there is any discrepancy in identification of mixed infections by multiple genotypes in paired clinical specimens obtained from patients and to determine the significance of viral load differences between patients infected with single and multiple genotypes. One hundred clinical specimens from 75 patients were included in the study. Real-time PCR; Multiplex PCR and PCR-based RFLP were applied for the determination of viral load and genotyping of HCMV, respectively. Out of the 75 patients, 36 (48%) carried multiple genotypes. Discrepancy with regard to detection of genotypes were found in 17/25 patients whose paired clinical specimens were analyzed. Mixed genotypes were found more often in peripheral blood than urine or intraocular fluids collected from the same patient. There was a statistically significant difference between the median viral loads of clinical specimens carrying single genotypes and multiple genotypes. Mixed infections with multiple genotypes were found predominantly in the leukocyte fraction of peripheral blood specimens. The detection of mixed infections by multiple genotypes in the hypervariable regions of HCMV can be a surrogate marker of an increase in viral load. J. Med. Virol. 81:861,869, 2009. © 2009 Wiley-Liss, Inc. [source] Detection of human bocavirus in respiratory, fecal, and blood samples by real-time PCR,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2009Sarah J. Tozer Abstract Human bocavirus (HBoV) has been detected worldwide in respiratory samples. Two real-time PCR assays, targeting the non-structural protein (NP-1) and viral protein (VP-1) genes, were designed and validated to detect HBoV in patients with respiratory disease, gastroenteritis, or systemic illness. Sensitivity of the NP-1 and VP-1 assays were equal to the conventional PCR assay previously described by Allander et al. [2005: Proc Natl Acad Sci USA 102: 12891,12896] being 100%, and giving specificity of 94% and 93%, respectively. There was no cross-reaction identified with unrelated respiratory agents, or to human DNA. The limits of detection were 10 copies of genomic DNA equivalents per reaction for both assays. The assays were used to screen three different sample populations, combined nose, and throat swabs (n,=,96) from children with acute respiratory disease, fecal samples (n,=,375) from adults, and children with gastroenteritis and whole blood (n,=,229) collected from 31 immunocompromised children taken over an 18-month period. In total 17 (18%) respiratory samples and 18 (4.8%) fecal samples were identified as having HBoV present. Of the pediatric whole blood specimens investigated, HBoV was detected in six (2.6%) samples from four patients. In summary, two real-time PCR assays targeting different genes were designed and validated for use as screening methods for the detection of HBoV. HBoV was found in three different specimen types: parent-collected combined nose,throat swabs, fecal samples collected from symptomatic individuals and whole blood from immunocompromised children. J. Med. Virol. 81:488,493, 2009. © 2009 Wiley-Liss, Inc. [source] HHV-6 and HHV-7 antigenemia related to CMV infection after liver transplantationJOURNAL OF MEDICAL VIROLOGY, Issue 6 2006Maiju Härmä Abstract Background Betaherpesviruses human herpesvirus-6 and -7 (HHV-6, HHV-7), which are closely related to cytomegalovirus (CMV), have been reported in transplant patients. In this retrospective study, we investigated the occurrence of HHV-6 and HHV-7 antigenemia in relation to symptomatic CMV infection after liver transplantation. Methods: Sample material from 64 adult liver transplant recipients was included in the study. The patients were monitored weekly for CMV, HHV-6, and HHV-7. CMV infections were diagnosed by pp65-antigenemia and viral cultures. Concomitantly HHV-6 and HHV-7 antigens were demonstrated in peripheral blood mononuclear cells by monoclonal antibodies against both variants A and B and immunoperoxidase staining. Altogether 540 post-transplant blood specimens were analyzed. Results: Nineteen patients (30%) developed symptomatic CMV pp65 antigenemia during the first 3 months (mean 33 days, range 5,62 days) post-transplantation and were treated with intravenous ganciclovir. Concurrent HHV-6 antigenemia was detected in 16/19 (median 9 days, range 6,24 days) and HHV-7 antigenemia 15/19 patients (median 17 days, range 5,58 days) after transplantation. HHV-6 appeared before CMV in most cases (12/16), HHV-7 usually together with CMV. In those cases that HHV-6 preceded CMV antigenemia, it also was a possible cause of graft dysfunction. HHV-7 and CMV were so closely overlapping, that no symptoms could solely be linked with HHV-7. Conclusion: HHV-6 and HHV-7 antigenemia usually occurred together with symptomatic CMV infection after liver transplantation. HHV-6 preceded CMV, but HHV-7 appeared together with CMV. Further investigation of the clinical significance of HHV-6 and HHV-7 antigenemia in organ transplant patients is necessary. J. Med. Virol. 78:800,805, 2006. © 2006 Wiley-Liss, Inc. [source] The epidemiology of venous thromboembolism in Caucasians and African-Americans: the GATE Study,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2003N. F. Dowling Summary., The aim of this study was to assess, comprehensively, medical and genetic attributes of venous thromboembolism (VTE) in a multiracial American population. The Genetic Attributes and Thrombosis Epidemiology (GATE) study is an ongoing case,control study in Atlanta, Georgia, designed to examine racial differences in VTE etiology and pathogenesis. Between 1998 and 2001, 370 inpatients with confirmed VTE, and 250 control subjects were enrolled. Data collected included blood specimens for DNA and plasma analysis and a medical lifestyle history questionnaire. Comparing VTE cases, cancer, recent surgery, and immobilization were more common in caucasian cases, while hypertension, diabetes, and kidney disease were more prevalent in African-American cases. Family history of VTE was reported with equal frequency by cases of both races (28,29%). Race-adjusted odds ratios for the associations of factor V Leiden and prothrombin G20210A mutations were 3.1 (1.5, 6.7) and 1.9 (0.8, 4.4), respectively. Using a larger external comparison group, the odds ratio for the prothrombin mutation among Caucasians was a statistically significant 2.5 (1.4, 4.3). A case-only analysis revealed a near significant interaction between the two mutations among Caucasians. We found that clinical characteristics of VTE patients differed across race groups. Family history of VTE was common in white and black patients, yet known genetic risk factors for VTE are rare in African-American populations. Our findings underscore the need to determine gene polymorphisms associated with VTE in African-Americans. [source] Molecular diagnosis of inheritable neuromuscular disorders.MUSCLE AND NERVE, Issue 4 2005Part I: Genetic determinants of inherited disease, their laboratory detection Abstract Understanding of the genetic basis of inheritable neuromuscular disorders has grown rapidly over the last decade, resulting in improved classification and understanding of their pathogenesis. A consequence of these advances has been the development of genetic tests of blood specimens for the diagnosis of many of these diseases. For many patients, these blood tests have eliminated the need for other more invasive diagnostic tests such as muscle or nerve biopsy, and for some patients, reduced exposure to immunosuppressive medication and its complications. The first part of this review focuses on the nature of genetic disorders, the laboratory methods used in the performance of genetic tests, and general practical aspects of their use and interpretation. The second part discusses the applicability of these tests to the range of neuromuscular disorders. Muscle Nerve, 2005 [source] Acute respiratory distress syndrome by cytomegalovirus infection in an immunocompetent infantPEDIATRIC PULMONOLOGY, Issue 8 2008Jungi Choi MD Abstract A 2-month-old female infant was admitted with progressive respiratory distress, fever, and diagnosed with acute respiratory distress syndrome (ARDS). The primary pulmonary pathogen was proven to be cytomegalovirus (CMV) from bronchoalveolar lavage fluid, urine, and blood specimens. Other immunologic findings were normal. CMV-induced ARDS has not been reported previously in immunocompetent infants. Pediatr Pulmonol. 2008; 43:824,827. © 2008 Wiley-Liss, Inc. [source] Increased Ig-null B lymphocytes in the peripheral blood of pediatric solid organ transplant recipients with elevated Epstein-Barr viral loadsPEDIATRIC TRANSPLANTATION, Issue 3 2009Elizabeth Schauer Abstract:, In this study, the characteristics of Ig-null B cells in high viral load carriers were examined by four-color flow cytometry. The frequency of Ig-null B cells in patients with high, low or undetectable virus loads was found that while patients with a high load had more Ig-null cells, these cells were also present in the low and undetectable load groups. As Ig-null cells from patients with no viral load were EBV-negative, EBV infection was not absolutely required for the generation or survival of Ig-null cells. Ig-null cells were CD19+, sIg - , CD5,, CD10,, CD27,, CD23,, CD38,, and CD69, with variable surface expression of CD20 and CD40. Ig-null cells did not have a proliferating cell phenotype (Ki67 - ) and a high proportion were HLA class I - and class II - . Virus copy number in CD19+ Ig-null cell populations may be much higher than in CD19+ Ig+ cell populations. EBV infected Ig-null cells were common in blood specimens from pediatric solid organ transplant recipients and infected Ig-null cells may pose potential problems for immunotherapies that target infected B cells directly. [source] Peripheral blood picture following mild head trauma in childrenPEDIATRICS INTERNATIONAL, Issue 3 2008Bulent Alioglu Abstract Background: The aim of the present study was to investigate changes in peripheral white blood cell, and differential counts following mild head trauma in a pediatric population. Methods: Fifty-one patients (mean age, 79 ± 62 months) with mild head trauma (Glasgow Coma Scale [GCS] score 15) who were admitted to the emergency department, were studied. Two blood specimens were collected from each patient, one on arrival and one after 24 h at the emergency department. Complete blood count was performed using a hemocytometer and the absolute cell counts for each sample were calculated after examination of peripheral smear. Results: No patient developed any complication during the hospital stay or after discharge. Significant differences were found for white blood cell, neutrophil, and immature cell counts just after and 24 h after trauma (P = 0.047, 0.039 and 0.009, respectively). Conclusions: Mild head trauma may cause an increase in white blood cell, neutrophil and band counts in children just after trauma. In a child with a mild head trauma, who is asymptomatic, with GCS score of 15 and absence of risk factors, and without clinical deterioration, complete blood cell count may be omitted from laboratory workup. But a prospective randomized study comparing mild head trauma patients with good and bad clinical outcome is needed to draw a definite conclusion. [source] Evaluation of a second-generation portable blood lead analyzer in an occupational settingAMERICAN JOURNAL OF INDUSTRIAL MEDICINE, Issue 12 2007Noel V. Stanton MS Abstract Background A new blood lead testing instrument has qualities that make the instrument attractive for on-site testing of occupational lead exposures. This study evaluated the accuracy of the instrument when used in a manufacturing setting, and examined the impact of blood storage and shipment on results. Methods Venous blood specimens (n,=,121) were obtained and immediately analyzed on-site using the new instrument. They were then shipped to a reference laboratory and analyzed using electro-thermal atomization atomic absorption spectrometry (ETAAS), and retested using the new instrument. Results The cohort blood lead concentration averaged 40.1 µg/dl. Results obtained on the new analyzer with freshly collected blood averaged 38.7 µg/dl. The mean difference of 1.2 µg/dl on paired samples was not statistically significant. Following blood shipment and storage, results on the analyzer increased to an average of 42.4 µg/dl. The mean increase of 3.0 µg/dl on stored blood samples also failed to reach statistical significance. Under OSHA proficiency test acceptability requirements, 94% of the results had satisfactory agreement. Conclusions The new analyzer might be a useful tool for on-site monitoring of occupational lead exposures. The manufacturer's instructions should be adhered to with respect to specimen age and storage requirements. Am. J. Ind. Med. 50:1018,1024, 2007. © 2007 Wiley-Liss, Inc. [source] Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor -buprenorphine in urine, blood and hair samplesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2006Donata Favretto A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor -buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d4 -buprenorphine (d4 -BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutralization prior to extraction. Enzymatic hydrolysis with , -glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1,×,150,mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH+ species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is definitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1,10,ng/mL in urine and blood, in the range 10,160,pg/mg in hair) and limits of detection of 0.05,ng/mL for both BUP and NBUP in blood and urine samples, of 4,pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death. Copyright © 2006 John Wiley & Sons, Ltd. [source] Cholesteryl Ester Transfer Protein (CETP) Genetic Variation and Early Onset of Non-Fatal Myocardial InfarctionANNALS OF HUMAN GENETICS, Issue 6 2008V. Meiner Summary Although Cholesteryl Ester Transfer Protein (CETP) mediates the transfer of cholesteryl esters and triglycerides between lipoprotein particles and thus plays a crucial role in reverse cholesterol transport, the association of variations in the CETP gene with acute myocardial infarction (MI) remains unclear. In this study we examined whether common genetic variation in the CETP gene is related to early-onset non-fatal MI risk in a population-based case-control study from western Washington State. Genotyping for the CETP ,2708 G/A, ,971 A/G, ,629 A/C, Intron-I TaqI G/A and exon-14 A/G (I405V) SNPs was performed in 578 cases with first acute non-fatal MI and in 666 demographically similar controls, free of clinical cardiovascular disease, identified randomly from the community. In-person interviews and non-fasting blood specimens provided data on coronary heart disease risk factors. In men, there was little evidence for an association between single SNPs and MI risk, but in women the age- and race-adjusted OR was found to be significant in 4 out of the 5 CETP single variants. Haplotype analysis revealed two haplotypes associated with MI risk among men. As compared to men homozygous for the most common haplotype D (,2708 G, ,971 G, ,629 C, TaqI G and exon-14 A), the fully-adjusted multiplicative model identified haplotype G (,2708 G, ,971 A, ,629 A, TaqI G and exon-14 G) was associated with a 4.0-6.0-fold increased risk of MI for each additional copy; [95%CI 2.4,14.8] and haplotype B (,2708 G, ,971 G, ,629 A, TaqI A and exon-14 A) showed a significant decreased risk for early onset MI [OR = 0.18; 95%CI 0.04 , 0.75]. An evolutionary-based haplotype analysis indicated that the two haplotypes associated with the MI risk are most evolutionarily divergent from the other haplotypes. Variation at the CETP gene locus is associated with the risk of early-onset non-fatal MI. This association was found to be independent of HDL-C levels. These data and the sex-specific findings require confirmation in other populations. [source] An association between chronic infection with Chlamydia pneumoniae and lung cancer.APMIS, Issue 9 2001A prospective 2-year study This study assesses a possible relationship between chronic Chlamydia pneumoniae (Cpn) infection and lung cancer (LC). A total of 210 consecutive patients (136 M, 74 F) were diagnosed with LC during a 2-year period. Blood was obtained from 128 M and 70 F patients for Cpn serology. Repeat blood specimens were taken after 3 months. Throat specimens for Cpn DNA analysis by PCR were taken from 110/136 M and 63/74 F. Seventy-four cytobrush specimens were taken and also analyzed by polymerase chain reaction (PCR). Fifty (29 M, 21 F) bronchial biopsies and 8 (6 M, 2 F) tumors resected at surgery were analyzed for Cpn by immunohistochemistry (IHC). Males had significantly more often squamous-cell carcinoma (SCC) than females. Other types of LC were more equally distributed between males and females. The difference between males and females regarding smoking history was significant, and male LC patients had significantly higher levels of IgG and/or IgA antibodies than female LC patients. Male and female LC patients had significantly higher prevalences of high antibody titers than controls. A high prevalence of unusually high titers of specific Cpn antibodies was found in male LC patients. This could indicate that LC may be induced by chronic Cpn infection, since stable high titers of Cpn antibodies, especially IgA, are a hallmark of chronic infections. [source] In vivo pharmacokinetics of ketoprofen after patch application in the Mexican hairless pigBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 4 2009Masafumi Horie Abstract To evaluate the pharmacokinetics of topical drugs, in vitro permeation studies are performed using sacrificed pig skin or human tissues resected at surgery; however, these methods have their limitations in in vivo pharmacokinetics. This study examined the usefulness of Mexican hairless pigs for in vivo pharmacokinetic study, especially the drug concentration in the tissues. A ketoprofen patch was applied on the back of Mexican hairless pigs for 24,h, followed by sequential collection of blood specimens from 0 to 36,h (n=3). Also, the skin, subcutaneous fat, fascia and muscle from the center of the site of application were excised at 12,h after the application (n=4). Ketoprofen was first detected in the plasma at 8,h, the concentration increasing up to 24,h; the plasma concentration began to decrease after the removal of the ketoprofen patch. Ketoprofen concentrations in the tissues decreased with increasing depth of the tissues, but the values in the deep muscles, being the lowest among the tissues examined, were still higher than those in the plasma. While the data of drug concentration in human tissue are difficult to test, the Mexican hairless pig model appears to be attractive for in vivo pharmacokinetic studies of topically applied ketoprofen. Copyright © 2009 John Wiley & Sons, Ltd. [source] | ||||||||||||||||