Aliquots Of Specimens Research Articles

Effects of Storage Temperature and Duration on Flow Cytometry Analysis of Bone Marrow Aspirate Samples

Abstract Introduction/Objective Multiparameter flow cytometry immunophenotyping is a powerful and indispensable tool in the diagnosis of hematologic malignancies, but its accuracy and precision are vulnerable to pre-analytical interferences. Acid-citrate-dextrose (ACD) is one of three main anticoagulants used for marrow collection. It offers better preservation and is commonly used for apheresis stem cell collection. However, ACD may alter the pH of short-draw samples. It is unclear how prolonged storage in ACD affects the results of flow cytometry testing. In this pre-analytical study, we characterized the effects of temperature and storage duration in bone marrow aspirate collected in ACD. Methods/Case Report Five bone marrow specimens were collected in ACD and stored at 4°C or room temperature (RT), respectively. Aliquots of specimens were tested at 24, 48, and 72 hours. A flow cytometry panel including CD19, CD33, CD34, CD38, CD45, CD138, and 7-AAD was used to identify lymphocytes, monocytes, granulocytes, myeloid blasts, hematogones, and plasma cells. Absolute cell count, percentage of leukocytes, percentage of membrane- compromised cells (7-AAD bright), and mean-fluorescent-intensities were measured for each subset. The results of each storage condition were compared to the results on the day of sample collection. Results (if a Case Study enter NA) We found that granulocytes and plasma cells are most vulnerable to delayed testing, especially when stored at RT. Myeloid blasts, hematogones, monocytes, and lymphocytes were largely stable within 48 hours at 4°C or RT, but their relative representation in total leukocytes was affected by the diminished granulocytes. Variations in surface marker expression of CD45, CD38, and CD138 in the plasma cell subset were also noticed. This effect was less profound at 4°C than RT. Conclusion We conclude that delayed testing of specimens collected in ACD within 72 hours does not significantly impact the results of flow cytometry testing for the commonly studied subsets, and 4°C may be a more robust and preferable method for storage.

Resistance of SARS-CoV-2 beta and gamma variants to plasma collected from Canadian blood donors during the spring of 2020.

BackgroundThis pilot study assesses the ability of plasma collected from Canadian blood donors in the first wave of the SARS‐CoV‐2 pandemic to neutralize later SARS‐CoV‐2 variants of concern (VOCs).Study design and methodsA repeated cross‐sectional design was used, and a random cross‐sectional sample of all available Canadian Blood Services retention samples (n = 1500/month) was drawn monthly for April and May of 2020. Qualitative IgG analysis was performed on aliquots of specimens using anti‐spike, anti‐receptor binding domain, and anti‐nucleocapsid protein enzyme‐linked immunosorbent assays as well as the Abbott Architect SARS CoV‐2 IgG assay (Abbott Laboratories) against the anti‐nucleocapsid protein. Selected plasma specimens were then assessed for neutralization against VOCs using pseudotyped lentivirus inhibition assays as well as plaque reduction neutralization test 50% (PRNT50).ResultsSix specimens with a high neutralizing titer against wild‐type SARS‐CoV‐2 and three specimens with a low neutralizing titer against wild‐type SARS‐CoV‐2 were chosen for further analysis against VOCs. Four of six high neutralizing titer specimens had a reduced neutralizing capacity against beta VOCs by both neutralization methods. Three of six high neutralizing titer specimens had reduced neutralization capacity against gamma VOCs.ConclusionsThis preliminary data can be used as a justification for limiting the use of first wave plasma products in upcoming clinical trials but cannot be used to speculate on general trends in the immunity of Canadian blood donors to SARS‐CoV‐2.

Anti-Apoptotic BCL-2 Family Members Confer Resistance to Calicheamicin-Based Antibody-Drug Conjugate Therapy of Acute Leukemia

BACKGROUND: With gemtuzumab ozogamicin (GO; targeting CD33) and inotuzumab ozogamicin (IO; targeting CD22), 2 antibody-drug conjugates delivering a toxic calicheamicin (CLM) derivative have recently been approved for the treatment of people with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), respectively. While effective in some, many patients do not benefit from these ADCs. It is unclear to what degree anti-apoptotic BCL-2 family members are involved in modulating efficacy of CLM-based ADCs, with limited studies coming to differing conclusions. Given the clinical availability of small molecule inhibitors for BCL-2 family proteins (BCLi), here we clarify the impact of BCL-2 family proteins on the anti-leukemic activity of CLM-ADCs. MATERIALS AND METHODS: Human AML and ALL cell lines were engineered to overexpress BCL-2, BCL-XL, and MCL-1 via lentiviral gene transfer. AML and ALL cell lines as well as AML patient samples were exposed to increasing concentrations of GO or IO with or without the BCL-2 inhibitor ABT-199 (venetoclax), the BCL-2/BCL-XL inhibitor ABT-263 (navitoclax), and the MCL-1 inhibitor AZD5991. Dead cells were enumerated by flow cytometry via 4',6-diamidino-2-phenylindole staining after 72 hours. For BH3 profiling of AML patient specimens, thawed AML patient specimen aliquots were exposed to JC-1 mitochondrial dye and BH3 peptides, and peptide-induced depolarization was then calculated as a percent relative to a CCCP positive control, yielding a priming score for each BH3 peptide. RESULTS: At a dose of 1000 pg/ml, GO killing of ML-1 (AML) cells decreased from 56±5% (mean±SEM) in parental cells to 32±7% (p<0.01) and 26±6% (p<0.01) in cells overexpressing BCL-2 and BCL-XL, respectively (all n=3). Similar results were seen in another AML cell line (HL-60). In REH (ALL) cells treated with IO, overexpression of BCL family members also reduced killing - at 500 pg/ml, 59±8% of cells were killed in contrast to 12±1% (p<0.01) of BCL-2-expressing and 11±1% (p<0.01) of BCL-XL-expressing cells, with similar results seen in another ALL cell line (RS4;11). Addition of ABT-199 or ABT-263 at 1 µM modestly increased GO-mediated killing of AML cell lines - for example, ML-1 cells treated with GO at 100 pg/ml, cytotoxicity increased from 41±6% to 57±7% (ABT-199, p<0.01) and 61±8% (ABT-263, p<0.01). The effect of BCLi was more pronounced on IO-mediated killing of ALL cell lines than on GO-mediated killing of AML lines. For example, killing of REH cells treated with IO at 25 pg/ml increased from 39±7% (without BCLi) to 72±8% (ABT-199 1 µM, p<0.01) and 87±9% (ABT-263 1 µM, p<0.01), with similar results seen in RS4;11 cells. BH3 peptide profiling of AML patient specimens treated with GO implicated MCL-1 as a potential additional modulator of AML response to GO. Consistent with this finding, overexpression of MCL-1 reduced leukemia cell death in HL-60 cells treated with GO (GO at 1000 pg/ml, 41±2 % vs. 26±1 %, p=0.01) and RS4;11 cells treated with IO (IO at 100 pg/ml, 76±2% vs. 27±6%, p<0.01). The MCL-1 inhibitor AZD5991 modestly increased the anti-leukemic efficacy of GO in ML-1 cells and AML patient specimens, but more dramatically enhanced IO killing of REH cells (IO at 10 pg/ml, 18±2% without AZD5991 vs. 70±2% with 0.1 µM AZD5991, p<0.01). The triplet combination of GO, ABT-199 and AZD5991 did not improve markedly on the ABT-199/AZD5991 combination in the absence of GO in cell lines or AML patient specimens, though the triplet combination of IO, ABT-199 and AZD5991 showed promising activity: in REH cells treated with 10 pg/ml IO, cytotoxicity was 18±2% without BCLi, 32±8% with ABT-199 0.1 µM, 19±2% with AZD5991 0.01 µM, and 56±14% with the triplet combination (p<0.01 for comparison of triplet combination with IO/BCLi doublet). CONCLUSIONS: Our studies establish an important role of anti-apoptotic BCL-2 family members as resistance factor for CLM-based ADC therapy of acute leukemia. These findings provide the rationale to explore the combination of small-molecule inhibitors of BCL-2 family members with CLM-ADCs as a combination strategy in the clinic to improve the efficacy of GO and, particularly, IO. These therapeutic strategies may incorporate the assessment of the relative contribution of specific BCL-2 family members to an individual cancer patient's disease. Disclosures Jean: Eutropics Pharmaceuticals: Employment. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership. Walter:Seattle Genetics: Research Funding; Kite Pharma: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy.

LC-MS-MS with Post-Column Reagent Addition for the Determination of Zolpidem and its Metabolite Zolpidem Phenyl-4-carboxylic Acid in Oral Fluid after a Single Dose.

A rapid, selective and sensitive LC-MS-MS method with a post-column addition of acetonitrile was developed and fully validated for the quantitative determination of zolpidem and its major metabolite, zolpidem phenyl-4-carboxylic acid (ZPCA), in oral fluid. Preliminary sample treatment was limited to a simple dilution of 1 mL oral fluid specimen aliquots with methanol. Chromatography was performed on a Capcell Pak C18 MGII column (250 × 2.0 mm, 5 μm i.d., Shiseido, Tokyo, Japan) with isocratic elution using a water-acetonitrile mobile phase with 0.1% formic acid, 5% acetonitrile and 20 mM ammonium acetate in an aqueous phase at a flow rate of 0.4 mL/min. Acetonitrile was added post-column at a flow rate of 0.4 mL/min to enhance ionization of the analytes in the MS source. Detection was carried out on a QTrapTM 6500 mass spectrometer in positive ionization mode. Good linearities were generated over the range of 0.05-200 ng/mL for zolpidem and 0.1-200 ng/mL for ZPCA. Limit of detection for zolpidem and ZPCA were 0.01 ng/mL and 0.05 ng/mL, respectively, whereas LLOQs were 0.05 ng/mL and 0.1 ng/mL, respectively. This method meets the required criteria for bioanalytical analyses to be used for clinical and forensic purposes. Application of this method was demonstrated by testing authentic samples collected after a single oral dose to obtain insights into the general detectability and detection windows of zolpidem and ZPCA in oral fluid.

Middle East respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in Amman, Jordan, March 2010 to September 2012

Hospitalized children < 2 years of age in Amman, Jordan, admitted for fever and/or respiratory symptoms, were tested for Middle East respiratory syndrome coronavirus (MERS-CoV): MERS-CoV by real-time RT-PCR (rRT-PCR). This was a prospective year-round viral surveillance study in children <2 years of age admitted with acute respiratory symptoms and/or fever from March 2010 to September 2012 and enrolled from a government-run hospital, Al-Bashir in Amman, Jordan. Clinical and demographic data, including antibiotic use, were collected. Combined nasal/throat swabs were collected, aliquoted, and frozen at –80°C. Specimen aliquots were shipped to Vanderbilt University and the Centers for Disease Control and Prevention (CDC), and tested by rRT-PCR for MERS-CoV. Of the 2433 subjects enrolled from 16 March 2010 to 10 September 2012, 2427 subjects had viral testing and clinical data. Of 1898 specimens prospectively tested for other viruses between 16 March 2010 and 18 March 2012, 474 samples did not have other common respiratory viruses detected. These samples were tested at CDC for MERS-CoV and all were negative by rRT-PCR for MERS-CoV. Of the remaining 531 samples, collected from 19 March 2012 to 10 September 2012 and tested at Vanderbilt, none were positive for MERS-CoV. Our negative findings from a large sample of young Jordanian children hospitalized with fever and/or respiratory symptoms suggest that MERS-CoV was not widely circulating in Amman, Jordan, during the 30-month period of prospective, active surveillance occurring before and after the first documented MERS-CoV outbreak in the Middle East region.

Laboratory diagnosis of tuberculosis in a large pediatric hospital in Cambodia

Tuberculosis laboratory in the Jayavarman VII Children's Hospital in Siem Reap, part of the Kantha Bopha Hospitals, the largest pediatric hospital complex in Cambodia. To determine the efficiency of on-site microscopy and rRNA amplification in children with a clinical diagnosis of tuberculosis (TB) and specimen sampling for culture. From 1 July 2005 to 31 March 2006, 52,400 children were admitted to the hospital. Among these, 405 children had tuberculosis, including 91 (22.5%) laboratory-confirmed cases, or respectively 7.7 and 1.7 per 1000 admissions. Among cases confirmed by microscopy or rRNA assay, rRNA identified 91.2%. Among all culture-confirmed cases, rRNA identified 90.5%. Culture alone contributed 7.1% to all laboratory confirmed cases. The yield of culture from preserved specimens was not affected by shipment delay. For 97.4% of the children, the maximum turnaround time for the on-site laboratory result was 48 h. Implementation of a mycobacteriology service in a referral hospital is feasible, as the molecular technique is highly efficient. Storage of specimen aliquots allows subsequent culture without loss of viability due to shipment delay.

Pyrosequencing Assay to Measure LINE-1 Methylation Level in Esophageal Squamous Cell Carcinoma

Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element 1 L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. LINE-1 methylation is a useful marker for predicting cancer prognosis and monitoring efficacy of adjuvant therapy. Nonetheless, no study has examined LINE-1 methylation in esophageal squamous cell carcinoma (ESCC). The aim of this study is to assess the precision of sodium bisulfite conversion and polymerase chain reaction (PCR) pyrosequencing assay for evaluating LINE-1 methylation in ESCC. To measure assay precision, we performed bisulfite conversion on 5 different DNA specimen aliquots (bisulfite-to-bisulfite) and repeated PCR pyrosequencing five times (run to run). Second, to assess heterogeneity of LINE-1 methylation levels within tumor, we made 5 different tissue sections from one tumor and examined LINE-1 methylation level of each section (section to section). Third, to evaluate LINE-1 methylation status in ESCC, we applied this assay to 30 ESCCs and 30 matched normal esophageal mucosa. Bisulfite-to-bisulfite standard deviation (SD) ranged from 1.44 to 2.90 (median 2.32) in ESCCs; and 0.57 to 4.02 (median 1.23) in normal esophagus. Run-to-run SD ranged from 0.63 to 3.25 (median 1.54) in ESCCs. Section-to-section SD ranged from 1.37 to 3.31 (median 1.94). ESCC tissues showed significantly lower levels of LINE-1 methylation than matched normal mucosa (P < .0001; n = 30). There was no significant relationship between LINE-1 methylation level and tumor stage (P = 0.14). Bisulfite conversion and PCR pyrosequencing assay can measure LINE-1 methylation in ESCC, and may be useful in clinical and research settings.

Validation of Neurokinin A Assays in the United States and Europe

International cooperative group trials require specific, sensitive biomarker assays that are validated between continents. Neurokinin A (NKA) has been shown to be a powerful independent predictor of a poor prognosis in well-differentiated midgut neuroendocrine tumors. We hypothesized that NKA concentrations of clinical specimens evaluated in NKA assays in the United States and the United Kingdom would be equivalent, even though assay techniques were significantly different. Frozen clinical specimen aliquots were shipped from the United States to the United Kingdom (n = 67), and from United Kingdom to the United States (n = 50). In addition, spiked plasma standards and medium-spiked standards were exchanged. Samples from the United States were directly assayed in a radioimmunoassay, whereas the UK specimens were extracted, and the reconstituted specimens assayed in the radioimmunoassay. Neurokinin A values from the 2 studies were analyzed by regression analysis. The NKA values from the US and UK laboratories were essentially identical (United States to United Kingdom, r = 0.88, P < 0.0001; and United Kingdom to United States, r = 0.96, P < 0.0001). Validation of biomarker assays across continents will ensure that laboratory observations made by researchers are equivalent and that prediction of clinical outcomes based on these assays is also reliable.

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen's kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.

Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells

Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.

Antineoplastic activity of idazoxan hydrochloride

PurposeIdazoxan hydrochloride (IDA) is a 241 molecular weight imidazoline and adrenoreceptor ligand. It binds to mitochondrial membranes and promotes apoptosis of pancreatic beta cells. Since IDA has not been tested against tumor cells, the purpose of our study was to determine if IDA has antineoplastic activity.MethodsWe used the conversion of a soluble tetrazolium salt to an insoluble formazan precipitate and differential staining cytotoxicity assays to determine if IDA was cytotoxic to cell lines of murine lung cancer and human prostate cancer, as well as to a variety of fresh human tumor samples. We used flow cytometry to analyze cell death and calreticulin expression.ResultsIDA is cytotoxic to both cell lines and against aliquots of specimens of breast, gastric, lung, ovarian and prostate cancers as well as non-Hodgkin’s lymphoma. It produces apoptotic cell death and promotes calreticulin expression, suggesting that IDA might be immunomodulatory in vivo.ConclusionWe anticipate that IDA will be clinically useful in cancer treatment.

Poor Performance of Universal Sample Processing Method for Diagnosis of Pulmonary Tuberculosis by Smear Microscopy and Culture in Uganda

Laboratory methods to improve smear microscopy are an urgent priority for global tuberculosis control. The novel universal sample processing (USP) method has been reported to improve conventional diagnostic testing for tuberculosis while also providing inhibitor-free specimens for molecular assays. However, no studies evaluating the method in the field have been conducted. In this study, we compared the performance of the USP method to that of the standard N-acetyl-L-cysteine-NaOH (NALC) method for conventional diagnosis of tuberculosis in 252 adults admitted to Mulago Hospital in Kampala, Uganda, with a clinical suspicion of pneumonia. A single early-morning sputum specimen collected from each patient was divided into two aliquots, each of which was assigned a random identification number. One randomly numbered specimen was processed by the USP method and the other by the NALC method. Mycobacterial cultures were more frequently negative in USP compared to NALC specimen aliquots (58% versus 43%; P < 0.001). There was no difference in the proportion of contaminated mycobacterial cultures (12% versus 11%; P = 0.87). The sensitivity and specificity of smear microscopy for the USP method were 52% and 86%, respectively, and were not significantly different from those for the NALC method (56% and 86%, respectively) using mycobacterial culture results as a reference standard. These results suggest that the USP method did not provide any significant advantage over the standard NALC method for conventional diagnosis of tuberculosis in our setting and illustrate the importance of well-designed, field-level evaluations of novel diagnostic techniques.

Lassa Fever, Nigeria, 2003 and 2004

Lassa Fever, Nigeria, 2003 and 2004

Use of real-time reverse transcriptase polymerase chain reaction assay and cell culture methods for detection of swine influenza A viruses

To evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay performed on pooled nasal swab specimens, compared with virus isolation performed on individual nasal swab specimens by use of 2 cell culture lines for detection of swine influenza A viruses. 900 nasal swab specimens obtained from pigs at an abattoir and 62 nasal swab specimens submitted for diagnostic testing. Primers were chosen to amplify a conserved portion of the influenza virus matrix gene. Assay sensitivity was initially determined by testing serial dilutions of various subtypes of swine influenza viruses. Sensitivity and specificity were confirmed by use of nasal swab specimens with or without addition of known concentrations of influenza virus and further validated by testing nasal swab specimens obtained through an abattoir surveillance program or submitted for diagnostic testing. Aliquots of specimens were pooled in sets of 10, and results of real-time RT-PCR assays were compared with results of virus isolation of individual specimens in Madin Darby canine kidney (MDCK) and mink lung (Mv1Lu) cells. Real-time RT-PCR assay was highly specific (100%) and sensitive (88% to 100%). Among the 16 viruses isolated, 3 grew only in Mv1Lu cells and 3 grew only in MDCK cells. Results indicate that real-time RT-PCR assay is a fast and accurate test for screening numerous nasal swab specimens for swine influenza virus. Some viruses were isolated in only MDCK or Mv1Lu cells, indicating that use of >1 cell line may be required to isolate a broad range of influenza A viruses.

Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens.

In the past decade, human immunodeficiency virus (HIV) lymphocyte immunophenotyping has evolved significantly. New fluorochromes, new multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using the single-platform technique have all contributed to the reliability of T-cell subset measurements. In this study, four gating protocols were evaluated to select the most robust method for T-cell subset enumeration. Peripheral blood specimens from 21 HIV(+) and 20 HIV(-) individuals were monitored up to 96 h. Aliquots of specimens were stored at room temperature and analyzed at 6 (baseline), 48, 72, and 96 h. Aliquots were stained with CD45-fluorescein isothiocyanate (FITC)/CD3PC5/CD4RD1/CD8ECD. Data analysis was performed with all four gating protocols. Only with fresh blood did all protocols provide similar results. From samples that were 48 h old, the choice of gating strategy had a dramatic impact on immunophenotyping results. The largest deviations from baseline values occurred at 96 h and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. The gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 h. By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days.

Use of alkaline phosphatase to correct the underestimation of fosphenytoin concentration in serum measured by phenytoin immunoassays.

The pharmacologic activity of fosphenytoin, a new phosphate ester pro-drug of phenytoin, is due to in vivo conversion to phenytoin. Fosphenytoin concentrations cannot be accurately estimated by phenytoin immunoassays (fluorescence polarization and chemiluminescence) owing to the nonlinear relation between fosphenytoin concentration and the observed cross-reactivity. The problem of slow conversion of fosphenytoin to phenytoin in serum in vitro can be circumvented by rapidly converting fosphenytoin to phenytoin in vitro by alkaline phosphatase. Drug-free serum, heparin, EDTA, or citrated plasma were supplemented with 2 concentrations of fosphenytoin. Then to 1-mL aliquots of specimen, no enzyme (control), 10 microL, or 25 microL of enzyme solution was added. The specimens were incubated, and phenytoin concentrations were measured by fluorescence polarization and chemiluminescent assays. In the absence of enzyme, we observed little conversion of fosphenytoin to phenytoin, but in the presence of only 10 microL of enzyme, the conversion of fosphenytoin to phenytoin was complete in 5 minutes. We also observed complete conversion of fosphenytoin to phenytoin by alkaline phosphatase in heparin, EDTA, and citrated plasma. If clinically indicated, the phenytoin concentration can be measured before and after addition of enzyme to roughly estimate the rate of conversion.

Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR.

A nested PCR for genus-specific amplification of the Chlamydia omp1 locus was established. This PCR detected single template molecules in 200-microl specimen aliquots. Amplified chlamydial omp1 alleles were typed by heminested species PCRs and allele PCRs. We applied this method to 407 specimens from several host animals with various clinical conditions, and we detected prevalences of chlamydiae from 6 to 50%. Amplicons from peacock enteritis and equine infertility specimens were not typeable according to present omp1 allelic criteria for the chlamydial species. DNA sequencing revealed novel omp1 alleles which were 29.9 and 47.6% divergent in the deduced peptide sequences from the most closely related chlamydiae. Phylogenetic reconstruction indicated segregation of these alleles from the current four chlamydial species (90 and 97% bootstrap support), thus strongly suggesting the existence of additional chlamydial species. Allele typing of amplicons from swine with intestinal, urogenital, and respiratory infections demonstrated several unique omp1 allelic variants of Chlamydia trachomatis. These novel alleles had deduced peptide sequences which were 11.6 to 19% divergent from porcine C. trachomatis S45. Mutations were clustered in the C-terminal region of variable segment IV of the omp1 locus encoding subspecies and serovar determinants of the chlamydial major outer membrane protein, thus implying that there are numerous serovars of porcine C. trachomatis. These results demonstrate the need for routine application of sensitive genus-specific detection of chlamydiae in animal specimens and suggest a more prominent role than anticipated for chlamydiae in animal diseases.

Biological characteristics of adenomatous nodules, adenomas, and hyperfunctioning nodules as defined by adenylate cyclase activity and TSH receptors

AbstractThis study was designed to examine the functional characteristics of thyroid adenomas, adenomatous (colloid) nodules, hyperfunctioning nodules, and normal thyroid parenchyma with respect to the presence of TSH receptors, levels of adenylate cyclase, and the responsiveness of adenylate cyclase to specific stimuli, e.g., NaF (sodium fluoride), GTPγS [guanosine 5′‐0‐(3‐thiotriphosphatate)], PGE1, and TSH. Aliquots of specimens from patients undergoing thyroidectomy [11 normal thyroids, 7 adenomas, 8 adenomatous (colloid) nodules, 5 hyperfunctioning nodules] were immersed in liquid nitrogen and stored at 90°C. Studies were performed on partially purified plasma membranes. Receptor binding of125I‐TSH was quantitated and analyzed according to Scatchard. Adenylate cyclase activity was assayed in the same tissue fraction by conversion of (α‐32P) ATP to cAMP.High‐ and low‐affinity TSH receptors were present in adenomatous (colloid) nodules, hyperfunctioning nodules, and normal thyroid parenchyma. The thyroid adenomas had high‐affinity receptors in normal quantities; low‐affinity receptors were present at normal or decreased levels. The basal adenylate cyclase activity was slightly increased in thyroid adenomas. All groups had adenylate cyclase response to NaF with the thyroid adenomas being 60% more responsive than normal thyroid parenchyma (p &lt;0.005). All tissues responded to PGE1, with normal thyroid parenchyma being twice as responsive as thyroid adenomas. The adenylate cyclase response of adenomatous (colloid) nodules and hyperfunctioning nodules was distinct from that of adenomas and comparable to that of normal thyroid parenchyma. The adenylate cyclase response to TSH was fourfold in thyroid adenomas and twofold in adenomatous (colloid) nodules compared to normal thyroid parenchyma.