Large Volumes Of Serum Research Articles

OR13-05 Longitudinal Changes In Bone Turnover Following Withdrawal Of RANKL Inhibition

Abstract Disclosure: A.S. Kim: None. A. Castro-Martinez: None. V. Taylor: None. J. Center: None. C.M. Girgis: None. P.I. Croucher: None. M.M. Mcdonald: None. Denosumab is an effective osteoporosis treatment, preventing bone loss by inhibiting RANKL. However, stopping denosumab leads to rebound bone mineral density(BMD) loss. This is due to accelerated bone resorption by osteoclasts. Serum bone turnover markers such as P1NP and CTX have been utilised in clinical practice to guide sequential therapy following denosumab discontinuation. However, an optimal strategy has not been established. Understanding the temporal changes in osteoclast activity will guide safe, effective sequential therapy following denosumab discontinuation. We hypothesised that serum TRAP5b, a marker of enzymatic activity of osteoclasts, would be a more useful marker in this context to guide sequential treatment following denosumab discontinuation. Seven-week-old female C57BL/6 mice were treated with 2-weeks of thrice-weekly OPG:Fc(10mg/kg) to inhibit RANKL then withdrawn from therapy (OPG-W) or saline(vehicle). Longitudinal BMD and serum TRAP5b were measured throughout the study. Mice were harvested at weeks 2, 8, 11 and 13 to allow a large volume of serum to be collected to concurrently measure serum TRAP5b and the products of collagen formation and breakdown, serum P1NP and CTX respectively . Following OPG:Fc withdrawal, BMD peaked at week 8 in OPG-W mice (92.94 vs75.09mg/cm2, p<0.0001), started to decline at week 10 and normalised to vehicle levels by week 13. Longitudinally, serum TRAP was suppressed by week 2 (0.05 vs 11.90U/L, p<0.0001) and remained suppressed until week 8, following which serum TRAP levels rose progressively to 64% above vehicle levels at week 12 (17.86 vs 10.92U/L, p<0.0001). The rise in TRAP between weeks 8 to 10 preceded the decline in BMD.Serum TRAP was significantly elevated in OPG-W mice at week 11 (15.45 vs 11.46U/L, p=0.01) whereas serum P1NP and CTX remained equivalent to vehicle levels. Serum TRAP, P1NP and CTX were significantly higher in OPG-W mice at week 13 by which time BMD had reached vehicle levels. Our findings show that rebound decline in BMD has already occurred by the time bone turnover markers used in clinical practice (P1NP and CTX) rise above vehicle levels. A significant overshoot in serum TRAP occurs earlier and prior to bone loss and may better inform sequential therapy following denosumab discontinuation. Presentation: Friday, June 16, 2023

ПОЛУЧЕНИЕ И ОЦЕНКА СПЕЦИФИЧНОСТИ ДИАГНОСТИЧЕСКОЙ СЫВОРОТКИ ДЛЯ ВЫЯВЛЕНИЯ СОСУДИСТОГО БАКТЕРИОЗА КРЕСТОЦВЕТНЫХ

Vascular bacteriosis of cruciferous is a dangerous disease of agricultural crops. It causes mass death of plants in the field, significant crop losses and contributes to its spoilage during storage, especially due to damage by other infections accompanying its development. There are no effective methods of combating its pathogen, therefore, an important condition for reducing the infection of cruciferous plants with this infection is its timely detection and prevention of the spread of infection. To date, this problem associated with the analysis of a large number of samples is most effectively solved by immunoassay methods, for the successful practical implementation of which a hyperimmune serum specific to the detected pathogen is necessary. The presented work is devoted to the creation of such a serum. Its preparation was carried out by immunization of rabbits with a preparation of disintegrated cell membranes of the causative agent of vascular bacteriosis of cruciferous – Xanthomonas campestris – using a 0.05% solution of polyazolidinammonium modified with hydrate ions of halogens as an adjuvant. As a result, it was found that the obtained serum is highly specific in relation to the detected pathogen. The use of the named adjuvant in its creation made it possible to achieve a level of sensitivity sufficient to exclude the influence on the analysis of microorganisms of other genera and species widely distributed in the wild. This makes it possible to use the serum effectively in diagnostic systems. Thanks to the use of another adjuvant, it was possible to reduce the time required for immunization of rabbits. This makes it possible to obtain large volumes of serum in a shorter time, which makes the analysis more accessible and allows it to be used more widely in everyday practice.

Predicting the presence and titre of rabies virus-neutralizing antibodies from low-volume serum samples in low-containment facilities.

Serology is a core component of the surveillance and management of viral zoonoses. Virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. Here, focusing on Rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmRFFIT) that overcomes these limitations. Specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein-tagged murine leukaemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. We further used statistical models to generate rapid, quantitative predictions of the probability and titre of rabies virus-neutralizing antibodies from microscopic imaging of neutralization outcomes. Using 47 serum samples from domestic dogs with neutralizing antibody titres estimated using the fluorescent antibody virus neutralization test (FAVN), pmRFFIT showed moderate sensitivity (78.79%) and high specificity (84.62%). Despite small conflicts, titre predictions were correlated across tests repeated on different dates both for dog samples (r=0.93) and in a second data set of sera from wild common vampire bats (r=0.72, N=41), indicating repeatability. Our test uses a starting volume of 3.5 µl of serum, estimates titres from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. The pmRFFIT enables high-throughput detection of rabies virus-neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained.

Development of a sensitive HPLC-MS/MS method for 25-hydroxyvitamin D2 and D3 measurement in capillary blood.

BackgroundMeasurement of 25‐hydroxyvitamin D [25(OH)D)] levels is important. The current method requires a relatively large volume of serum. To minimize the amount of serum needed, we established a high‐performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) method to measure 25(OH)D in capillary serum.MethodsVenous blood and fingertip blood were collected from 90 participants. Volumes of 100 µL of venous serum and 20 µL of capillary serum were collected. The serum samples were pretreated by protein removal, extraction and concentration, and an HPLC‐MS/MS method based on chromatographic separation and multi reactive ion monitoring was conducted. The intra‐ and inter‐batch variation coefficients were less than 10% for both 25‐hydroxyvitamin D3[25(OH)D3] and 25‐hydroxyvitamin D2[25(OH)D2)]. For venous specimens, the accuracies were 3.87% and 4.91%, respectively. For capillary specimens, the accuracies were 1.65% and 5.32%, respectively.ResultsThe limit of detection (LOD) of 25(OH)D3 was 0.01 ng/mL, and the LOD of 25(OH)D2 was 0.05 ng/mL. The results showed that the mean concentration of 25(OH)D in venous blood was 22.56 ± 9.50 ng/mL, while the mean concentration of 25(OH)D in capillary blood was 18.14 ± 7.86 ng/mL. Furthermore, the adjusted capillary blood 25(OH)D level was 22.99 ± 10.24 ng/mL by the correction formula in our study. Similarly, the mean concentration of 25(OH)D3 in capillary blood was 17.98 ± 7.98 ng/mL. The adjusted capillary blood 25(OH)D3 level was 22.85 ± 10.42 ng/mL. No difference in the content of 25(OH)D or 25(OH)D3 was found between venous serum and corrected capillary serum. The correlation coefficients between venous and corrected capillary concentrations of 25(OH)D and 25(OH)D3 were 0.7941 and 0.8103, respectively, and the areas under the receiver operating characteristic curve were 0.9367 and 0.9565, respectively.ConclusionsThis capillary blood method requires minimal sample preparation and is suitable for routine use in the 25(OH)D detection.

Fabrication of Microarrays for the Analysis of Serological Antibody Isotypes against Food Antigens.

Food intolerance is delayed adverse food reactions which follow consumption of specific foods. The underlying mechanisms are not well understood, but food intolerance is often considered as a type 2 hypersensitivity reaction mediated by immunoglobulin G (IgG) antibody. To understand the causes of food intolerance, it is important to investigate sensitization patterns of food-specific IgGs (sIgG) in relation to dietary patterns and physical conditions. Conventional approaches to measure serological IgGs often require large volumes of serum, thus are not suitable for highly multiplexed assays. To overcome this impracticality, we developed a highly sensitive method to screen the sIgGs and other antibody isotypes against 66 antigens with minimal amount of serums. We prepared a microarray by immobilizing food antigens on activated glass slides. Human sera and their dietary information were obtained from 30 subjects. Aliquots (200 nl) of sera were analyzed against 66 food antigens in parallel. sIgG levels were determined and analyzed in relation to subjects’ dietary patterns. The levels of antibody isotypes were also examined to understand the relationship between allergy and food intolerance. The developed microarray showed exceptional performances in antibody screening and demonstrated the potential to be used as an automated assay system.

Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS.

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5pg/mL while using only 0.175mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6 × 50mm, 1.8μm, 600bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5mL per minute. The calibration curve was found to be linear over a range of 5-10,000pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20pg/mL), medium (1000pg/mL), and high (5000pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ± 15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5months at - 70°C), and post-preparative storage (for 48h).

‘Insulin autoantibody affinity measurement using a single concentration of unlabelled insulin competitor discriminates risk in relatives of patients with type 1 diabetes

Development of high-risk combinations of multiple islet autoantibodies and type 1 diabetes is associated with high-affinity insulin autoantibodies (IAA), but IAA affinity measurements require large serum volumes. We therefore investigated whether a simplified method of IAA affinity measurement using a low concentration of unlabelled insulin (ULI) competitor discriminated between moderate-high- and low-affinity IAA and identified individuals at highest risk of disease. Samples were assayed by radiobinding microassay using high (4·0 × 10(-5) mol/l) and low (7 × 10(-9) mol/l) ULI concentrations for competitive displacement in three cohorts of IAA-positive individuals; (1) 68 patients with newly-diagnosed type 1 diabetes; (2) 40 healthy schoolchildren; and (3) 114 relatives of patients with type 1 diabetes followed prospectively for disease development (median follow-up 13 years). IAA results obtained with low ULI were expressed as a percentage of those obtained with high ULI and this was used to classify samples as low or moderate-high affinity (0-50% and >50%, respectively). Sixty-eight patient samples were positive with high and 67 (99%) with low ULI. Forty schoolchildren were IAA-positive with high and 22 (55%) with low ULI (P < 0·001). Of the relatives, 113 were positive with high and 83 (73%) with low ULI (P < 0·001). In relatives, moderate-high affinity IAA were associated with multiple islet antibodies (P < 0·001) and greater diabetes risk than low affinity IAA (P < 0·001). A single low concentration of ULI competitor can act as a surrogate for complex IAA affinity measurements and identifies those IAA-positive relatives at highest risk of disease progression.

Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA > IgM >>>>> IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.

Development of a focus reduction neutralization test (FRNT) for detection of mumps virus neutralizing antibodies

Although the plaque reduction neutralization test (PRNT) is considered the “gold-standard” assay for measuring neutralizing antibodies for mumps, it is technically demanding, slow and requires large serum volumes, which limits its use for investigating mumps vaccine efficacy and population susceptibility. Therefore, an immunocolourimetric-based focus reduction neutralization test (FRNT) was developed and validated against PRNT using 30 blood donor plasma samples (16 positive, 5 equivocal, and 9 negative for mumps IgG by EIA). The samples were tested in triplicate by FRNT and PRNT in 10 and 4 separate assay runs, respectively, and 50% neutralizing antibody titres calculated using the Kärber formula. There was good correlation between the two neutralization assays ( R 2 = 0.88). Inter-assay variation for FRNT titres was 2-fold, compared to a 3-fold variation for PRNT titres. From the distribution of results, a positive cut-off for FRNT was defined as 1:4. In conclusion, FRNT has similar sensitivity to the PRNT and offers the advantage of speed (2 days vs. 7 days), reduced sample volume (40 μL vs. 150 μL), and the possibility of automation using 96-well plates. FRNT appears to be a good substitute for PRNT for characterising the immune response to mumps and for vaccine efficacy studies.

Detection of CirculatingAspergillus fumigatusDNA by Real-Time PCR Assay of Large Serum Volumes Improves Early Diagnosis of Invasive Aspergillosis in High-Risk Adult Patients under Hematologic Surveillance

Detection of galactomannan antigen (GMA) in serum is the standard assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematological disorders. Detection of Aspergillus DNA in serum has been proposed, but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. In a 13-month prospective study, we compared the performances of twice-weekly screening of serum for GMA by an enzyme immunoassay and weekly screening for Aspergillus fumigatus DNA by a real-time PCR (RT-PCR) assay of 1.0 ml (LSV) or 100 mul (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC (European Organization for Research and Treatment of Cancer)/MSG (Mycoses Study Group)-documented IA. In all, 1,870 samples were screened for GMA. The sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV, respectively) of GMA for IA were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 938 samples for Aspergillus DNA by using LSV; 404 of these samples were also tested with SSV. The Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100%, respectively, with LSV and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, with SSV. DNA detection gave a positive result when performed on LSV in two cases of IA where the GMA assay result remained negative. Furthermore, in four IA cases, DNA was detected earlier than GMA. The use of LSV for extraction improved the performance of the RT-PCR, which appears highly sensitive and specific for the early diagnosis of IA in high-risk patients with hematological disorders.

Multi-laboratory simulator studies on effects of serum proteins on PTFE cup wear

A multi-laboratory, simulator study investigated the wear of polytetrafluorethylene (PTFE) cups run in bovine serum. Each laboratory used its own test protocol with a variety of simulator types. Our wear model incorporated 32mm dia CoCr heads matched to PTFE cups run with serum protein-concentrations in the range 17–69mg/ml. The multi-lab data demonstrated that protein-concentration had the most significant effect on wear performance. Both inverted and anatomical cups followed the same trend with first a rapid increase in wear-rates apparent for the initially low-protein levels and then a wear-rate reduction effect becoming apparent beyond 17mg/ml of proteins. The results showed that as the protein concentration increased from 17 to 69mg/ml, the magnitude of the wear-rates increased 200% but the protein wear-rate gradient decreased 24–60% with “inverted” and “anatomical” cups, respectively. This effect was more pronounced with ‘anatomical” than “inverted” cups. Thus, the wear-trends with “inverted” cups were generally the more consistent, particularly at the low-protein levels. Increasing the serum volume by two-fold in one study increased the PTFE wear-magnitudes approximately 40% and the protein-wear gradient by 30%. These PTFE wear phenomena were consistent with the concept that low-concentrations of proteins promoted polymer wear but high-protein concentrations resulted in a protein-degradation phenomenon which progressively masked the actual polymer wear. In the selected protein range 17–69mg/l, the multi-laboratory simulator data consistently overestimated the average clinical wear-rate by at least 50–100% depending on protein range. It would, therefore, appear clinically relevant to study PTFE wear with an inverted-cup model using a large volume of serum but only in low-protein concentrations. The protein-related wear phenomena observed with PTFE cups in this multi-laboratory project may also have relevance for wear-simulation of UHMWPE cups.

Nutritional Management of Neonatal and Infant Liver Disease

After completing this article, readers should be able to: 1. Describe the caloric requirements of a neonate who has chronic liver disease. 2. Identify the vitamin and mineral deficiencies associated with cholestasis and the methods used to correct these deficits. 3. Describe the relationship between total parenteral nutrition and the development of chronic liver disease. 4. Delineate the relationship between malnutrition at the time of liver transplantation and subsequent morbidity and mortality. 5. List the best measurements of nutrition assessment in infants who have chronic liver disease. The liver is involved in many of the body’s metabolic processes, including: regulation of protein, fat, and carbohydrate metabolism; vitamin storage and activation; and detoxification and excretion of waste products. The healthy liver synthesizes and excretes bile salts. With cholestatic liver diseases, poor bile flow results in fat malabsorption because micelles, which solubilize long-chain fatty acids, cannot form without bile salts. Fat malabsorption results in enteric losses of the fat-soluble vitamins A, D, E, and K. Impaired liver function can lead to nutrient deficiencies and eventually protein-energy malnutrition. All of these factors, combined with the common symptoms of anorexia and poor dietary intake, make malnutrition common in chronic liver disease, particularly if it occurs in infants, who are more vulnerable to the debilitating effects of malnutrition because of their higher energy and growth requirements. A wide range of deficits occurs in most chronic liver diseases of children (Table 1⇓ ). Malnutrition itself may induce further derangements of liver function because the liver requires energy for a number of synthetic, storage, and detoxification functions. View this table: Table 1. Nutritional Risk Factors in Acute and Chronic Liver Disease The need for aggressive, early nutrition support in neonates has been well established and discussed extensively. Some centers routinely use a “standardized” approach to nutrition support; we use a “tailored” approach that entails parenteral or …

Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treatment may predict sustained and relapse response in chronic HCV infection

The ability to predict accurately a sustained response during therapy in patients with hepatitis C virus (HCV) infection is unavailable. The aim of this study was to differentiate, during therapy, patients who would relapse from those with a sustained response by ultracentrifugation for residual serum HCV RNA. Sixty-one specimens (from 32 patients) collected during interferon therapy were assessed by ultracentrifugation. All were negative using a quantitative polymerase chain reaction (PCR) (detection limit < or = 100 copies/ml). One-milliliter aliquots were ultracentrifuged at 23,000 x g (160 min), and then the nucleic acid pellet was extracted, precipitated, and resuspended. Qualitative PCR was carried out in quadruplicate using two separate 5'UTR primer sets (8 results/specimen). A specimen was positive if > or = 1 gels was positive compared to controls. At weeks 12 and 24, 9/9 (100%) sustained response patients were negative by ultracentrifugation. In the 23 relapse patients at week 12, 7/12 specimens were positive; at week 24, 7/14 were positive. Earlier time points could not differentiate the patients' eventual response to therapy. The predictive value of a positive ultracentrifugation test for relapse at week 12 or 24 was 100%. The predictive value of a negative test for sustained response was 62% and 50% at week 12 and 24, respectively. These preliminary results indicate that patients with an eventual sustained response will have no detectable serum HCV RNA by week 12 or week 24. A positive result is 100% predictive of relapse.

Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treatment may predict sustained and relapse response in chronic HCV infection

The ability to predict accurately a sustained response during therapy in patients with hepatitis C virus (HCV) infection is unavailable. The aim of this study was to differentiate, during therapy, patients who would relapse from those with a sustained response by ultracentrifugation for residual serum HCV RNA. Sixty-one specimens (from 32 patients) collected during interferon therapy were assessed by ultracentrifugation. All were negative using a quantitative polymerase chain reaction (PCR) (detection limit ≤ 100 copies/ml). One-milliliter aliquots were ultracentrifuged at 23,000 × g (160 min), and then the nucleic acid pellet was extracted, precipitated, and resuspended. Qualitative PCR was carried out in quadruplicate using two separate 5′UTR primer sets (8 results/specimen). A specimen was positive if ≥ 1 gels was positive compared to controls. At weeks 12 and 24, 9/9 (100%) sustained response patients were negative by ultracentrifugation. In the 23 relapse patients at week 12, 7/12 specimens were positive; at week 24, 7/14 were positive. Earlier time points could not differentiate the patients' eventual response to therapy. The predictive value of a positive ultracentrifugation test for relapse at week 12 or 24 was 100%. The predictive value of a negative test for sustained response was 62% and 50% at week 12 and 24, respectively. These preliminary results indicate that patients with an eventual sustained response will have no detectable serum HCV RNA by week 12 or week 24. A positive result is 100% predictive of relapse. J. Med. Virol. 57:351–355, 1999. © 1999 Wiley-Liss, Inc.

Evaluation of Homogeneous High-Density Lipoprotein Cholesterol Assay on a BM/Hitachi 747–200 Analyzer

Because of the inverse correlation that exists between serum HDL-C concentration and risk of atherosclerotic disease (1), monitoring of high-density lipoprotein cholesterol (HDL-C) in serum is clinically important. In 1993, the National Cholesterol Education Program Adult Treatment Panel II (NCEP ATP II) revised its guidelines for diagnosis and treatment of hypercholesterolemia in adults to include HDL-C measurement at the initial screening stage when total cholesterol is measured (2). The enhanced role of HDL-C in clinical practice increases the need for reliable and readily performable HDL-C measurement. Most routine laboratories use a two-step method: chemical precipitation of lipoproteins containing apoprotein B, then quantification of HDL as cholesterol remaining in the supernate. Such precipitation-based methods are time-consuming, labor-intensive, require relatively large volumes of serum, and cannot be fully automated. Recently, several methods of direct HDL-C measurement have become commercially available, including the use of magnetically responsive particles with polyanion-metal combinations (3), the use of polyethylene glycol (PEG) with antibodies against apoprotein B and apoprotein CIII (4)(5), and the use of PEG-modified enzymes and sulfated α-cyclodextrin (6)(7). Okazaki et al. (8) also recently proposed the use of high-performance liquid chromatography (HPLC) as a tool to compare different methods for HDL-C. The goal of this study was to evaluate three assays for homogeneous HDL-C on BM/Hitachi 747–200 Automatic Analyzer (Boehringer Mannheim Corp.): the direct HDL-cholesterol reagent kit (Boehringer Mannheim Corp.), the N-geneousTM HDL cholesterol reagent kit (Genzyme Diagnostics), and EZ-HDLTM cholesterol reagent kit (Sigma Diagnostics). For each HDL-C assay, the reagent kit contains Reagent 1 and Reagent 2; Reagent 1 is liquid, and Reagent 2 is lyophilized. Reaction principles of these three methods are quite different; the direct-HDL uses PEG-modified cholesterol esterase and cholesterol oxidase as well as sulfated α-cyclodextrin to provide selective determination of HDL-C in serum; …

High-performance liquid chromatography of haloperidol in serum at the concentrations achieved during chronic therapy.

Abstract Haloperidol, 4-[4-( p -chlorophenyl)-4-hydroxypiperidino]-4′-fluorobutyrophenone, is a widely prescribed neuroleptic which is used in both adults and children for the treatment of acute and chronic psychotic syndromes and abnormal movements. Therapeutic monitoring of haloperidol is indicated for the following reasons; (1) there is no direct relationship between daily doses and serum levels at steady state; (2) saturation kinetics may apply at higher doses; (3) commonly associated drugs or disease states may alter the drugs pharmacokinetic parameters; (4) drug disposition is faster in children than adults; (5) optimum serum concentrations vary between pathological syndromes and age groups, and (6) side effects and adverse reactions are clearly related to haloperidol serum concentrations [1]. To date all the accepted methods for monitoring haloperidol have used gas chromatography (GC) and large volumes of serum [2]. A recent electron-capture GC method required only 1 ml of plasma or serum, but the selectivity of the assay was not reported [3]. The present method overcomes the use of such high sample volumes, is simpler to perform and is of equal or higher sensitivity. It has been used to monitor haloperidol serum concentrations in various psychiatric patients and the results of these determinations in 28 patients are presented.

A Novel Micro-assay for Insulin Autoantibodies

Insulin autoantibodies (IAA) are established markers of Type 1 diabetes and are widely used for the prediction of this disease. Standard assays require relatively large serum volumes for reliable measurement of IAA, limiting their use in young children. We have developed a novel small volume assay which is suitable for screening large numbers of samples. For reasons of economy we have adopted a two-stage strategy in which all samples are screened for insulin binding and those with raised levels are quantified in an assay using competitive displacement. Using this assay 126 out of 241 (52%) newly diagnosed IDDM patients (median age 10.2, range 1.3–20.7 years) had IAA levels above the 99th centile of 2860 schoolchildren (median age 11.3, range 9.0–13.8 years), including 81 out of 117 (69%) patients below the age of 10 years. The assay compared well overall when measuring IAA in direct comparison with a conventional assay. We conclude that reliable measurement of IAA is possible on less than 50μl of serum using this novel assay and that this should facilitate large scale screening, particularly in young children.

Use of serum from patients with polycythaemia or haemochromatosis for laboratory external quality assurance exercises.

Some External Quality Assessment Schemes (EQAS) require large volumes of human serum. During a one year period, 595 units of blood were obtained from 87 patients with haemochromatosis and polycythaemia, who underwent therapeutic venesection at the Edinburgh and South East Scotland Blood Transfusion Service. Serum from 59% of these donations was used in the EQAS for peptide hormones and related substances. The cost of the serum collection was 109 pounds/litre, but was only 33 pounds/litre of serum if the cost of the actual venesection was excluded. Results from tests on the sera were satisfactory in a variety of immunoassays for several different hormones. EQA schemes with requirements for large volumes of serum should consider therapeutic venesection as a cost effective means of obtaining serum.

Detection limits of organochlorine pesticides and related compounds in blood serum

Determinations of organochlorine pesticides and similar chemical residues in blood serum have often reported detection limits of 1 ng/mL. When a study group has incurred body burdens lower than this, underestimates and misclassifications of exposure may occur because persons with pesticide residue concentration below the limit of detection are usually treated as zeros.' Thus in order to more accurately assess exposures in such populations, analysis of adipose tissue has been done. Recently, with TCDD, use of a sufficient volume of serum, as much as 0.5 L, in conjunction with appropriate analytical techniques has been shown to achieve detection limits necessary for epidemiological assessments, i.e., comparable to analysis of adipose tissue. In a population-based study involving children in which the authors were involved, it was not feasible to obtain specimens of either adipose or a large volume of serum. There was no compelling health motivation for such measures, nor did they wish to impair participation rates. Therefore, they chose to optimize the existing serum analysis, in order to achieve a detection limit low enough to assess reasonably the anticipated exposures.

Possibilities of Bacteremia and Toxemia in Death of Chickens Infected with Eimeria tenella

The possibility that bacteremia and toxemia were the causes of death in cases of cecal coccidiosis was investigated. Germ-free and ordinary chickens with microflora were inoculated with sporulated oocysts of Eimeria tenella. At 5 days postinoculation, cecal lesions in ordinary chickens were more severe than those in germ-free ones. Cardiac blood, spleen, and liver were examined in ordinary chickens for bacteremia and endotoxemia, and small numbers of bacteria were recovered from both infected and uninfected birds. Endotoxin levels in plasma of E. tenella-infected birds were low and not different from the levels of uninfected controls. To examine unknown toxic factors, a large volume of serum from infected chickens was injected intravenously into uninfected birds. No significant clinical signs were observed. It is concluded that the intestinal bacteria increase the severity of coccidial lesions without bacteremia and toxemia.