Dilution Of Specimens Research Articles

Seroprevalence of Getah virus in Pigs in Eastern China Determined with a Recombinant E2 Protein-Based Indirect ELISA.

Getah virus (GETV), in the genus Alphavirus and the family Togaviridae, has been detected throughout the world. GETV causes high morbidity and mortality in newborn piglets, entailing serious economic losses. Therefore, the experimental work on GETV detection is necessary. However, due to the influence of a variety of unavoidable factors, the ELISA test for the primary screening of animal diseases has low accuracy in detection results. Therefore, we optimized a recombinant E2 (rE2) protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of GETV antibodies in pig serum. The E2 protein was successfully expressed and purified with SDS-PAGE. A Western blotting analysis of sera from infected pigs showed strong reaction with a viral antigen of ~46 KDa corresponding to the E2 glycoproteins. By using chessboard titration and comparing the P/N values, we found that the optimal concentration of coated antigen was found to be 24.5 μg/mL, and the optimal dilution of serum specimens was 1:100. The best working dilution of the horseradish peroxidase (HRP)-conjugated goat anti-pig immunoglobulin (IgG) was 1:5000. The optimal coating conditions were 12 h at 4 °C. The optimal incubation conditions for serum specimens, blocking, and reaction with the secondary antibody were all 1 h at 37 °C. We also investigated the seroprevalence of GETV in 133 serum specimens collected in Eastern China, and 37.59% of the samples tested positive for anti-GETV IgG antibodies, indicating that the seroprevalence of GETV is high in pig populations in China. The seroprevalence was significantly lower in spring (April; 24.24%, 16/66) than in autumn (October; 50.75%, 34/67), which suggested that the presence of anti-GETV antibodies in pigs was seasonal. In conclusion, we improved an rE2 ELISA that detected pig antibodies against GETV after experimental and natural infections. This should be useful in the diagnosis and surveillance of GETV infections.

Antibody-Free Quantification of Serum Chromogranin A by Targeted Mass Spectrometry.

Chromogranin A (CgA) is a 48 kDa protein that serves as a diagnostically sensitive, but nonspecific, serum biomarker for neuroendocrine tumors. Immunoassays for CgA are not standardized and have a narrow dynamic range, which requires dilution of concentrated specimens. We developed and validated an antibody-free, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for CgA without these limitations. CgA was extracted from serum using a mixed-mode anion exchange solid-phase extraction plate, digested with trypsin, and analyzed by LC-MS/MS using well-characterized CgA calibration standards. After validation, the mass spectrometry method was compared with the CISBIO immunoassay using 200 serum specimens previously submitted for CgA analysis. Specimens with discordant results were reanalyzed by high-resolution mass spectrometry- (HRMS) -based methods to assess the contribution of truncated and post-translationally modified forms of CgA. The assay had a linear range of 50 to 50 000 ng/mL, recoveries between 89% and 115%, and intra- and interassay imprecision <10%. LC-MS/MS assay results showed a Pearson's correlation of r = 0.953 with the CISBIO immunoassay, with CgA values being a mean 2- to 4-fold higher. Concordance for CgA between the 2 assays was 80.9% (95% CI 72.8%-89.2%), showing substantial agreement. Truncation and posttranslational modification, including 2 phosphorylation sites that had not been previously observed or predicted to our knowledge, did not appear to contribute directly to discordance between the 2 assays. Quantification of CgA by LC-MS/MS provides an analytically sensitive and reproducible alternative to commercially available immunoassays.

DEVELOPMENT AND APPLICATION OF A RAPID SCREENING METHOD FOR DETERMINATION OF NEW PSYCHOACTIVE SUBSTANCES AND THEIR METABOLITES IN URINE

The ongoing appearance of new psychoactive substances on the black market of illegal drugs, as well as the lack of information on their influence on the human body, faces several challenges in their determination by standard analytical techniques. Moreover, the rapid metabolism of new psychoactive substances reveals in the absence of possibility in the identification of their native structures in biological fluids. This study presents a new screening method for determination 137 psychoactive substances including their metabolites. 'Dilute-and-shoot' method was chosen as the preferable sample preparation technique, and consisted of 1:5 dilution of urine specimens with the solution of acetonitrile and water (30:70) followed by electrospray ionization – liquid chromatography-tandem mass spectrometry analysis. The developed qualitative method was validated according to United Nations Office on Drugs and Crime requirements that included assessment of selectivity, limits of detection, precision, and stability. In addition, the presented method was tested on 50 certified positive urine specimens containing different drugs of abuse. The confirmatory analysis was performed using a high-resolution mass-spectrometry approach. The presented screening method provides the possibility of simultaneous determination of synthetic cannabinoids (96), opioid analgesics (16), stimulators (13), hallucinogens (5), benzodiazepines (5) and non-classified drugs (10) during one run. The validation assessments of the novel method have shown high rates of its specificity, selectivity, intra- and inter-day precision and stability with the limit of detection ranged from 1 to 5 ng?mL-1. At the same time, tests of 50 positive samples showed excellent applicability of the developed screening method for routine preliminary screening analysis in toxicological laboratories.

An approach to analytical validation and testing of body fluid assays for the automated clinical laboratory

BackgroundBiochemical analysis of body fluids may lend insight into the pathogenesis of disease. Most commercially-available clinical laboratory methods are intended for blood-derived specimens and/or urine and laboratories must characterize the analytical performance of these methods for other specimen types such as body fluids. The aim of this work is to demonstrate one approach for characterizing analytical performance of assays for clinical analysis of body fluids. MethodsResidual waste samples were obtained from clinically ordered testing. Validation studies were performed for 8 chemistry analytes on Roche Cobas 6000 c501 (Roche Diagnostics, Inc.) analyzers. Accuracy, precision, analytical measurable range, reportable range, analytic sensitivity, interferences, analyte stability were assessed. Laboratory workflow for body fluid handling was designed based on results obtained. ResultsSample matrix interferences were not observed for the body fluids tested and assay reportable ranges used for serum were validated for body fluids. Dilution of body fluid specimens with saline demonstrated non-linear recovery of some enzyme activities. Assay imprecision was comparable to the manufacturer's claims for serum. The serum index thresholds for interference from hemoglobin and lipemia were lower for body fluids compared to manufacturer's stated limits for serum. Pretreatment of body fluid samples with hyaluronidase caused a 28% false increase in lipase activity, and 13% increase in total protein concentration. Ambient temperature analyte stability in body fluids was ≤24 h for most analytes compared to manufacturer's stated stability of 7 days for serum. In contrast to serum, lactate dehydrogenase (LDH) was labile in frozen body fluid specimens. ConclusionsValidation of analytical methods for body fluid testing is a necessary exercise to exclude potential matrix effects and set pre-analytic specimen quality criteria.

Evaluation of dilution and normalization strategies to correct for urinary output in HPLC-HRTOFMS metabolomics.

Reliable identification of features distinguishing biological groups of interest in urinary metabolite fingerprints requires the control of total metabolite abundance, which may vary significantly as the kidneys adjust the excretion of water and solutes to meet the homeostatic needs of the body. Failure to account for such variation may lead to misclassification and accumulation of missing data in case of less concentrated urine specimens. Here, different pre- and post-acquisition methods of normalization were compared systematically for their ability to recover features from liquid chromatography-mass spectrometry metabolite fingerprints of urine that allow distinction between patients with chronic kidney disease and healthy controls. Methods of normalization that were employed prior to analysis included dilution of urine specimens to either a fixed creatinine concentration or osmolality value. Post-acquisition normalization methods applied to chromatograms of 1:4 diluted urine specimens comprised normalization to creatinine, osmolality, and sum of all integrals. Dilution of urine specimens to a fixed creatinine concentration resulted not only in the least number of missing values, but it was also the only method allowing the unambiguous classification of urine specimens from healthy and diseased individuals. The robustness of classification could be confirmed for two independent patient cohorts of chronic kidney disease patients and yielded a shared set of 49 discriminant metabolite features. Graphical Abstract Dilution to a uniform creatinine concentration across urine specimens yields more comparable urinary metabolite fingerprints.

A case of Langerhans cell histiocytosis following acute basophilic leukemia

Dear Editor, Langerhans cell histiocytosis (LCH) is a class of histiocytic cell neoplasm with a clonal neoplastic proliferation of Langerhans-type cells that express CD1a, langerin, and S100 protein and show evidence of Birbeck granules by ultrastructural examination [1]. Concurrent LCH and malignancy have been reported occasionally. LCH occurred before or after malignancies including acute lymphoblastic leukemia, acute myeloid leukemia, and several solid tumors such as malignant lymphoma, retinoblastoma, lung carcinoma, ependymoma, hepatocellular carcinoma, Askin tumor, etc. [2–5]. We present the case of a 38-year-old patient who was diagnosed with LCH following acute basophilic leukemia (ABL). A 38-year-old male presented to the emergency department with a 2-month history of indigestion, nausea, and easy fatigue. Complete blood count with differential showed a white blood cell count of 13,500/μL with 13 % blasts and 14 % basophils, 8.4 g/dL hemoglobin, and a platelet count of 110,000/μL. Hepatosplenomegaly and multiple lymphadenopathies were noted. Bone marrow (BM) aspirates showed 15.6 % leukemic blasts and 11.6 % basophils. Since marked dilution of BM aspirate specimens, the proportion of blasts and basophiles were inaccurate. However, the BM cellularity was approximately 100 % with leukemic blasts. The blasts were of medium-to-large size, had round or cleaved nuclei, contained one or two nucleoli, and a had small amount of bluish cytoplasm. Some blasts had basophilic granules (Fig. 1). Flow cytometric analysis confirmed the presence of the surface markers CD34, CD13, CD33, CD117, and CD65. The blasts showed negative reaction for myeloperoxidase, periodic acid stain, and nonspecific esterase but metachromatic positivity after staining with toluidine blue. Electron microscopy confirmed the presence of basophilic granules in blasts (Fig. 1). The karyotype analysis demonstrated trisomy 16 (47,XY,+16[3]/46,XY[17]). The patient was diagnosed with ABL and received induction chemotherapy. Three months after induction chemotherapy, multiple erythematous indurated patches were observed on the left lower abdomen. Skin biopsy showed evidence of diffuse neutrophilic infiltration with papillary dermal edema and eczematous lesions, without evidence of cancer or fungal infection. Follow-up of BM studies revealed a normocellular marrow with 4.0 % blasts. However, marked increase of histiocytic cells, including immature forms, was noted. The histiocytic cells were clustered in a nodular pattern on the BM clot section. They were of medium-to-large size, with grooved or lobulated nuclei, inconspicuous nucleoli, and moderate amount of homogeneous pinkish cytoplasm. The histiocytic cells were positive for CD1a, S100, and CD68 marker proteins by immunohistochemistry staining (Fig. 2). Based on the morphology and immunohistochemical staining profiles, the patient was diagnosed with BM involvement of LCH. However, a primary lesion of LCH was not found. The patient underwent haploidentical BM transplantation; however, his clinical course continued to deteriorate with systemic fungal infection, necrotizing pneumonia, and hemodynamic instability. Despite aggressive treatment, the patient expired 7 months after diagnosis of ABL. T.-D. Jeong : S. Jang (*) :C.-J. Park :H.-S. Chi Department of Laboratory Medicine, College of Medicine, University of Ulsan and Asan Medical Center, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, South Korea e-mail: ssjang@amc.seoul.kr

Evaluation of Blood Agar Microtiter Plates for Culturing Leishmania Parasites To Titrate Parasite Burden in Spleen and Peripheral Blood of Patients with Visceral Leishmaniasis

Serial dilution of blood and spleen biopsy specimens, plated on Novy-MacNeal-Nicolle (NNN) blood agar using microtiter culture plates, is a sensitive and reproducible method for detection and growth of Leishmania parasites. Plates could be easily monitored, and growth could be rapidly detected. Moreover, parasite number may be estimated using this technique.

Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses

The use of flow cytometric enumeration of blasts in bone marrow aspirates has been of limited value in situations where blood contamination of the specimen is present. Assessment of sequential pulls of bone marrow aspirates from the same patient show decreasing proportions of blasts that are detected in later specimens. To address this problem, the intensity of CD16 on maturing neutrophils was compared for bone marrow biopsies, peripheral blood, and bone marrow aspirates. A comparison between bone marrow biopsy and aspirate specimens from the same individuals showed similar proportions of neutrophils with mature phenotype in most, but not all pairs. Other cell populations (total mature lymphocytes, monocytes, neutrophils, and blasts) were also similar between the two specimen types, with one exception of a patient with myelodysplasia, exhibiting a unique blast population in the biopsy that was not evident in the aspirate. The proportion of mature myeloid cells expressing a mature neutrophil phenotype (high levels of CD16) was found to be 17% (±6.7, n=47) in trephine marrow biopsy specimens. In contrast, marrow aspirates contained more of the mature neutrophil phenotype (38%±16, n=33) with about 1/3 of the aspirates indistinguishable from biopsies. Using a simple formula to normalize the aspirate specimens to the average neutrophil composition of marrow biopsies, it was possible to correct for the dilutional effect of added blood to both normal bone marrow aspirates and aspirates with elevated blast counts. These results suggest three alternative means of circumventing the problem of blood dilution of marrow aspirate specimens. (1) Blast counts by flow cytometry can be obtained from disaggregated biopsy specimens. (2) A bone marrow aspirate can be assessed for the proportion of mature neutrophils present and only those with low proportions (<30%) of phenotypic mature neutrophils are considered adequate for blast counting. (3) The aspirates with high proportions of mature neutrophils may be normalized based on the proportion of dim CD16 maturing myeloid cells to a level observed in bone marrow biopsies (based on an average mature neutrophil composition). Such an approach for identifying the amount of hemodilution in each specimen may enhance the utility of flow cytometry in enumeration of blasts in bone marrow, especially in cases where myeloblast count is crucial for prognosis, such as myelodysplastic syndromes (MDS).

Detection of Pathogenic Protozoa in the Diagnostic Laboratory: Result Reproducibility, Specimen Pooling, and Competency Assessment

Stool microscopy as performed in clinical parasitology laboratories is a complex procedure with subjective interpretation. Quality assurance (QA) programs often emphasize proficiency testing as an assessment tool. We describe a result reproducibility assessment tool, which can form part of a broader QA program, and which is based on the blinded resubmission of selected clinical samples, using concordance between the reports of the initial and resubmitted specimen as an indicator. Specimens preserved in sodium acetate-acetic acid-formalin can be stored for several months for use in such a program. The presence of multiple protozoa in one specimen does not affect concordance. Some dilution of specimens occurs in this process, and this may explain poor concordance when specimens with low protozoal concentrations are resubmitted. Evaluation of this tool in a large parasitology laboratory revealed concordance rates for pathogenic protozoa (Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Dientamoeba fragilis) of about 80%, which may be considered for use as a benchmark value. We also used this tool to demonstrate that when pairs of specimens from one patient are pooled to create a single specimen, concordance between the results of the individual and pooled specimens is high.

HBV DNA Quantitation Using Real-time PCR

Accurate measurement of the concentration of hepatitis B virus (HBV) DNA in clinical samples is important for the appropriate treatment of patients and evaluation of their therapeutic responses. In addition, the concentration of HBV DNA in the serum of patients with chronic HBV infection has a very broad range. Real-time PCR is very sensitive and useful to detect HBV DNA in a wide range of concentrations. We designed new primers and probes for real-time PCR to detect HBV DNA. Primers and probes specific for HBV were designed. EUROHEP HBV DNA standards (NIBSC, Hertfordshire, UK) with the HBV DNA concentration of 7.0 x 10(4) copies/mL was used to determine the calibration curve and efficacy for the real-time PCR assay. Sensitivity, dynamic range, and precision were evaluated. The correlation between the real-time PCR and Cobas Amplicor HBV Monitor(TM) assay in the measurement of serum HBV DNA concentrations in 52 patients with chronic HBV infection was evaluated. The sensitivity of the assay was approximately 6.08 x 10(2) copies/mL for HBV, and the quantitation was accurate and reproducible over a wide dynamic range from 6.1 x 0(2) to 6.5 x 10(9) copies/mL without any dilution of specimens. The assay showed low coefficients of variation of repeatability (3.7-24.9%) and reproducibility (7.8-24.7%). The results were found to correlate well with those obtained by Cobas Amplicor HBV Monitor(TM) kit. We provide a new in-house method for the measurement of serum HBV DNA using real-time PCR, which enables us to detect HBV DNA rapidly, sensitively, and accurately.

An investigation into the extent of possible dilution of specimens received for urinary drugs of abuse screening

Recent American and Swedish studies have shown an increase in "false" negative results when analysing dilute urine specimens for drugs of abuse. In the light of these studies, it was decided to perform a pilot study to determine the extent of possible specimen adulteration and dilution in a random batch of 50 urine specimens presented to this laboratory, using creatinine, osmolality, pH and relative density. It was found that 20% of the specimens were outside the pH range associated with the optimum working of Syva EMIT Drugs of Abuse in Urine (DAU) immunoassay screening techniques, and that if the National Institute on Drug Abuse (NIDA) recognized dilution cut-off of 1.8 mmol/L for urine creatinine concentration is applied, 84% of the specimens surveyed here would need to be repeated to ensure accurate results. Because of these findings, it is recommended that routine creatinine and pH estimations should be performed on all specimens submitted for urinary drugs of abuse screening, or at least when unexpectedly negative results are obtained.

Modification of Rapid Human Immunodeficiency Virus (HIV) Antibody Assay Protocols for Detecting Recent HIV Seroconversion

Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score=0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A 11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A 11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics=0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A 11-LS SOD values (R(2)>0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA.

Free secretory component as a standardization protein for nasopharyngeal specimens from children with upper respiratory tract infection

Free secretory component (FSC) has been recommended as a reliable protein for correction of the unknown dilution in tracheal aspirate samples from preterm infants. To investigate whether FSC would also provide a valid standardization protein for samples of nasopharyngeal secretions, this study determined the intersubject variation and the alteration over time in the concentrations of FSC in nasal secretions from 35 children (median age 14 months) who participated in an antibiotic efficacy trial. Nasopharyngeal aspirates were obtained at enrolment and after 2–3 d. FSC in the specimens was quantified by a direct enzyme immunoassay. The concentrations of FSC in the nasal secretions ranged from 0.08 to 189.6 μgml−1 (median 12.3 μgml−1); the ratio of the highest to the lowest concentrations was 2370, the difference between the 90th and 10th percentile concentrations was 189‐fold and the difference between the 75th and 25th percentile values was 26. FSC concentrations were significantly lower in children aged &lt;12 months (median 2.2 μg ml−1) than in the older children (median 21.5 μg ml−1; p= 0.035). Between the first and the follow‐up specimens, 65% of the children had &gt;2‐fold difference in the levels of FSC in the secretions. Because an optimal standardization protein should show minimal variation between individuals and over time, FSC may not be a suitable protein for correction of the unknown dilution of nasopharyngeal specimens from children with upper respiratory tract infection.

An investigation into the extent of possible dilution of specimens received for urinary drugs of abuse screening

An investigation into the extent of possible dilution of specimens received for urinary drugs of abuse screening

Serological detection of HBeAg and anti-HBe using automated microparticle enzyme immunoassays

Fully automated microparticle enzyme immunoassays (EIA) were developed for the detection of HBeAg (IMx HBe) and antibodies against HBeAg (IMx anti-HBe), respectively. Specimens from blood donors, diagnostic and hospital patients and individuals with a variety of infectious and immune diseases were tested both in house and at four clinical sites. The overall agreement between IMx HBe and Abbott HBe RIA/EIA was 99.7% (2985 of 2994) and between IMx anti-HBe and anti-HBe RIA/EIA was 95.8% (2330 of 2432). Almost all anti-HBe discordant specimens (94.1%, 96 of 102) were reactive by IMx anti-HBe but negative by anti-HBe RIA/EIA. All anti-HBe discordant specimens were also reactive for anti-HBc. The IMx anti-HBe assay was 2- to 4-fold more sensitive than the current RIA as determined by serial dilution of anti-HBe reactive specimens. The ability of these IMx assays to detect HBeAg and anti-HBe in 199 HBsAg reactive specimens was also evaluated. 43.7% (87 of 199) and 66.3% (132 of 199) specimens were reactive for HBeAg and anti-HBe by IMx, respectively. Only one specimen was negative for both IMx assays compared to 14 (7.0%) non-reactive for both HBe and anti-HBe RIA. There were 24 specimens (12.1%) positive for both HBeAg and anti-HBe by IMx compared to 1 (0.5%) positive by the corresponding RIAs. This increased detectability of anti-HBe in HBsAg carriers using IMx anti-HBe may result from increased sensitivity for ‘free’ anti-HBe and/or increased ability to detect anti-HBe in immune complex. IMx anti-HBe also detected more reactives among volunteer blood donor specimens reactive for anti-HBc but negative for HBsAg (55.5%, 86 of 155), compared to RIA (38.7%, 60 of 155). IMx anti-HBe may be useful in confirming prior exposure to HBV in blood screened positive by Corzyme.

Rapid screening for bacteriuria in pregnancy

We evaluated a bioluminescence assay as a screening test for the detection of bacteriuria in pregnancy. A total of 1,000 urine specimens from a randomly selected group of pregnant women undergoing prenatal surveillance was investigated. Sequential dilution of urine specimens on CLED agar plates served as a reference method. Set against the reference group, bioluminescence screening scored a 93% sensitivity, a 78% specificity and a 99% predictive accuracy for negative results. All urine specimens were also analysed chemically for the presence of nitrite and leucocyte esterase by dip sticks. Dip sticks proved to be insufficient because of poor sensitivities of 54% and 59%, respectively. The bioluminescence assay is an effective, time- as well as labor-saving but questionably cost-effective method for the detection of bacteriuria in pregnancy.

Plasma adrenocorticotropin in domestic geese: Effects of ether stress and endocrine manipulations

A direct radioimmunoassay for the determination of avian adrenocorticotropin (ACTH) in a small volume of plasma was developed using an antiserum specific for N-terminal region of the ACTH molecule. The sensitivity of the two stage assay is 0.1 fmol ACTH/tube. The specificity of the antiserum was tested by its cross reactions with synthetic ACTH fragments and by comparing curves obtained by dilution of different plasma specimens to that of ACTH 1–39 reference standard. Adrenocorticotropin responses of chronically cannulated geese to ether stress were evaluated and compared to changes of plasma corticosterone (B) concentration over a 2-hr period. ACTH showed a maximum between 5 and 10 min after ether exposure, while B peak appeared 10–15 min later. Thirty minutes after ether inhalation plasma ACTH returned to the baseline, while B response was longer-lasting and decreased to the resting level between 60 and 120 min. Basal and stress-induced ACTH plasma levels were also investigated in male and female gonadectomized and thyroidectomized geese. Castration increased, while thyroidectomy decreased the basal ACTH concentration. These endocrine manipulations did not, however, markedly affect the stress-induced ACTH hypersecretion except in thyroidectomized ganders, where the increment of plasma ACTH 10 min after ether inhalation was significantly lower than in sham operated control geese.

Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool.

A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed specimens; the increase averaged 1,336%. The assay was specific for antigens of this organism and for antigens derived from the cyst, as opposed to the trophozoite, stage. The assay could detect the antigens of five cysts per well, but could not detect antigen in in vitro-cultured trophozoites. A mouse monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which was prepared against cysts of G. lamblia, was used as the solid-phase capture antibody. The antibody was reactive with the cyst wall, as determined by immunofluorescence. Polyclonal rabbit anti-cyst IgG was used as the secondary antibody, and peroxidase-labeled goat anti-rabbit IgG was used as the tertiary antibody in the assay format. Maximal capture of antigen from stool specimens occurred by 30 min. Optimal dilution of specimens was in the range of 1:60 to 1:600. Preliminary characterization of affinity-purified antigen recognized by the monoclonal antibody showed that it is heat stable (100 degrees C, 12 min) and resistant to sodium periodate treatment and that it may exist in multiple molecular weights from 45,000 to 110,000.

Modification of the EMIT tox benzodiazepine assay for screening of alprazolam in serum.

Alprazolam (Xanax) is a relatively new triazolobenzodiazepine which is anxiolytic in man and is also prescribed in the treatment of panic attacks. The objective of this study was to optimize the reactivity of the EMIT tox serum benzodiazepine assay for toxicologic screening of alprazolam. The standard EMIT procedure was extensively modified so that therapeutic concentrations of alprazolam could be detected. Modifications for Methods A and B included a single dilution of specimens, increasing incubation temperature to 37 degrees C, dilution of Reagents A and B, and increasing specimen volume to 100 microL. Method B also included supplementing Reagent A with additional glucose-6-phosphate and beta-nicotinamide adenine dinucleotide. Using serum standards, the modified EMIT assays have detection limits of approximately 25 ng/mL (Method A) and 20 ng/mL (Method B). The patient specimens analyzed were considered positive by Method B and 24/28 positive by Method A. All 28 patient specimens tested by Method B were confirmed positive by gas chromatography/electron capture detection. The day-to-day precision of both methods was less than 2% CV with diluted EMIT calibrators (N = 6). In conclusion, the modified EMIT assay can be used to screen for alprazolam in serum at therapeutic concentrations.

Suspected periodontopathic microorganisms and their oral habitats in young children.

Samples of subgingival plaque from 67 children, 5-7 years of age, were examined for the presence of certain suspected periodontal pathogenic species using the conventional technique of anaerobic sonification, dilution and spiral plating. When this technique was compared with a direct plating procedure which involved no preliminary dispersion and dilution of plaque specimens, it was found that the direct method resulted in double the frequency of children in whom black-pigmented Bacteroides (BPB) were detected and a 10-times increase in the number of subjects harbouring Actinobacillus actinomycetemcomitans. Samples from the tongue, tonsils and saliva were also plated using the direct technique. BPB were detected less commonly in the plaque specimens (61.3% of children) than in saliva (89.5%), or on the tongue (86.6%) and tonsils (97.1%). Expressed as percentages of a pooled sample of the total BPB population, the most frequently detected species in plaque were Bacteroides intermedius (44.4%) and Bacteroides melaninogenicus (48.0%). The most prevalent isolate in all other oral sites was B. melaninogenicus. Expressed as percentages of children in whom BPB were detected, the most frequently isolated species from plaque using the conventional dilution technique was B. intermedius (21.3%), whereas other BPB species were present in fewer than 5% of children. Fusobacterium nucleatum and Capnocytophaga species were isolated most frequently from plaque but were also commonly detected in the various other oral sites.