Novel application of internal obturator and semitendinosus muscle flaps for rectal wall repair or reinforcement.

Internal obturator and/or semitendinosus muscle flaps were used to reinforce primary appositional rectal wall repair in three dogs and one cat in this case series. All three dogs incurred rectal wall compromise during surgical excision of anal sac tumours. The cat sustained bite wounds to the perianal region resulting in abscessation and a rectal tear. Our results indicate that application of an internal obturator and/or semitendinosus muscle flap can reduce the risk of rectal wall dehiscence after primary repair, and consequently the risk of pararectal abscess or rectocutaneous fistula formation.

Ureteral ectopia and urethral sphincter mechanism incompetence: an update on diagnosis and management options.

The two leading causes of urinary incontinence in dogs are ureteral ectopia in juveniles and urethral sphincter mechanism incompetence in adults. While the accuracy of diagnosis of ectopic ureters has improved due to increased use of CT and/or cystoscopy, the diagnosis of urethral sphincter mechanism incompetence largely remains one of exclusion. New treatment options have been developed for both conditions, which have reduced morbidity and mortality, although the rate of long-term urinary continence has not significantly improved for either and neither has our understanding of the pathophysiology behind these failures. This review provides updates on the management of both of these conditions, with discussion of controversial areas and thoughts for future directions.

Is calibration the cause of variation in liquid chromatography tandem mass spectrometry testosterone measurement?

BACKGROUND: Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of testosterone. Variation is seen between assays and this may be due to differences in calibration as commercial calibrators for this assay are not readily available. We investigated the effects calibration in routine clinical LC-MS/MS assays. METHODS: All LC-MS/MS users that were registered with the UKNEQAS external quality assurance scheme for testosterone were invited to take part in the study. A set of seven serum samples and serum-based calibrators were sent to all laboratories that expressed an interest. The laboratories were instructed to analyse all samples using there own calibrators and return the results and a method questionnaire for analysis. RESULTS: Fifteen laboratories took part in the study. There was no consensus on supplier of testosterone or matrix for the preparation of calibrators and all were prepared in-house. Also, a wide variety of mass spectrometers, internal standards, chromatography conditions and sample extractions were used. The variation in results did not improve when the results were corrected with a common calibrator. CONCLUSIONS: The variation in results obtained could not be attributed to variations in calibrators. The differences in methodologies between laboratories must be the reason for this variation.

A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solid-phase extraction.

BACKGROUND: The plasma renin activity (PRA) assay measures the ability of renin to generate angiotensin I (AngI) from angiotensinogen. It is used to monitor mineralocorticoid therapy and to screen hypertensive individuals for primary aldosteronism (PA). METHODS: Samples were incubated in the presence of protease inhibitors for 6.5 and 24 h. The reaction was stopped by the addition of 2% ammonium hydroxide. AngI was then quantified by liquid chromatography tandem mass spectrometry using online solid-phase extraction (XLC-MS/MS). RESULTS: This method requires a sample volume of 50 µL and has an inter-assay precision <14% across the working range. A 6.5-h incubation gave a lower limit of quantification (LLOQ) of 0.3 nmol/L/h and this can be reduced to 0.08 nmol/L/h using a 24-h incubation. Comparison to a radioimmunoassay revealed excellent correlation (r(2) = 0.98), but a 37% negative bias. We also found that renin is stable in whole blood for up to 24 h at room temperature. In contrast, storage at 4°C should be avoided as prorenin cryoactivation can affect the PRA result in some patient groups. CONCLUSIONS: We have developed and fully validated a semi-automated XLC-MS/MS method for the measurement of PRA. In addition, a reference range specific to this assay has been defined. We have also demonstrated that renin is stable for up to 24 h at room temperature. This will enable this assay to be extended to samples taken in primary care, potentially increasing the number of hypertensive patients who can be screened for PA.

Testosterone measurement by liquid chromatography tandem mass spectrometry: the importance of internal standard choice.

BACKGROUND: Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of testosterone. Variation is seen between assays and is likely to be due to method differences. One area of inconsistency among assays is the choice of internal standard. We investigated the effects of three internal standards. METHODS: Testosterone with two deuterium (D2), five deuterium (D5) and three carbon 13 enrichment (C13) were separately assessed. Samples were extracted using ether following the addition of 10 µL of internal standard. All aliquots were prepared in triplicate, one for each type of internal standard. After mixing, the ether was transferred to a 96-deep well block, and then evaporated to dryness. Extracts were reconstituted with 50% mobile phases and analysed using a Waters Acquity UPLC and Quattro Premier tandem mass spectrometer. This method had previously been shown to have excellent agreement with a reference method using the D2 internal standard and this was considered the target. RESULTS: Lower results were obtained when using D5 testosterone when compared with D2 testosterone. The C13 internal standard also gave lower results, but was closer to the D2 target than the D5 internal standard. CONCLUSIONS: The choice of internal standard alone can have a significant affect on the results obtained by LC-MS/MS assays for testosterone using this chromatography. The effects of the combination of chromatography and internal standard choice should be investigated during method development.

Unilateral hydronephrosis and hydroureter secondary to ureteric atresia, and uterus unicornis in a young terrier.

A nine-month-old entire female terrier cross was presented with intermittent anorexia, vomiting and recent onset of abdominal pain and distension. A diagnosis of unilateral hydronephrosis was made following ultrasound examination and intravenous urography, but no cause was identified. Subsequent ureteronephrectomy and histology of the affected kidney showed ureteric atresia as the cause of obstruction. Uterus unicornis was also identified and ovariohysterectomy was performed. The combination of structural abnormalities can be explained by an in-utero developmental error of their common embryological precursor, the mesonephric duct.

Cortisol measurement in patients receiving metyrapone therapy.

Current guidance recommends titrating the dose of metyrapone against serum cortisol concentration, in patients under medical management of Cushing's syndrome. In the UK, this almost always involves measuring serum cortisol concentration by immunoassay, the performance of which is questionable in the presence of altered steroid metabolism. Sera from two patients receiving metyrapone were analysed using a liquid chromatography tandem mass spectrometry (MS) steroid assay to identify which steroids, if any, were elevated in these patients. In addition, control serum was spiked with a series of steroids to identify any potential positive interferences in a cortisol immunoassay. Serum 11-deoxycortisol concentration was elevated in both of the patients studied. One patient also had an elevated serum 17-hydroxyprogesterone concentration and the other an elevated androstenedione. In addition, the results of the interference studies indicated that the cortisol immunoassay was susceptible to interference from 11-deoxycortisol, 17-hydroxyprogesterone and 21-deoxycortisol. However, the magnitude of interference, in the serum cortisol immunoassay, due to these three steroids could not account for the discrepancy between the cortisol concentrations measured by immunoassay and those measured by MS. Both clinicians and laboratory staff should be aware of these interferences when monitoring patients undergoing treatment with metyrapone, and consequently serum should be measured in these patients by MS, not by immunoassay.

A simplified liquid chromatography tandem mass spectrometry assay, using on-line solid-phase extraction, for the quantitation of cortisol in saliva and comparison with a routine DELFIA method.

BACKGROUND: We have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring salivary cortisol, which requires only 200 microL sample and no extraction. METHODS: Sample (200 microL) and 25 microL internal standard were added directly to a 96-deep-well plate. Of this, 50 microL was loaded onto a guard cartridge, the cartridge was then washed and the eluate was diverted to waste. The compounds were eluted from the guard cartridge onto the C18 analytical column. Cortisol and deuterated cortisol were monitored using transitions m/z 363.2 > 121.1 and 365.1 > 122.2, respectively. RESULTS: The method had a lower limit of quantitation of 2 nmol/L. Intra-assay and inter-assay imprecision were better than 9.5%. Comparison with an established dissociation enhanced lanthanide fluorescent immunoassay (DELFIA) gave good agreement for the majority of samples; LC-MS/MS = 1.0065 x DELFIA - 3.7 (n = 130). The reference range was determined to be 5.8-45.7 nmol/L at 08:00 h and <6.4 nmol/L at 23:00 h (n = 44). CONCLUSIONS: We have developed a simple, robust assay to measure salivary cortisol using on-line solid-phase extraction to reduce sample clean-up requirements.

Development of a method to measure plasma and whole blood choline by liquid chromatography tandem mass spectrometry.

BACKGROUND: Current gold standard markers for myocardial damage are troponins I and T, which are both sensitive and specific for the detection of myocardial infarction, but require up to 6 h to become reliably elevated in serum. Investigation into markers with potential to identify patients with early ischaemic changes is therefore intense. Choline is reported to be prognostic in patients presenting with acute coronary syndromes via its release from ischaemic cell membranes. METHODS: Liquid chromatography tandem mass spectrometry was used to develop a method to quantitate choline in plasma and blood. The method involves addition of a deuterated internal standard to an aliquot of plasma or blood followed by organic solvent addition, which precipitates the proteins in the sample. Preparation was carried out directly into a 96-deep-well plate. Chromatography of choline used a strong cation exchange column and separation used a Waters Atlantis dC18 analytical column positioned directly before the mass spectrometer source, allowing on-line preanalytical clean up of the sample. RESULTS: The lower limit of quantitation was 0.38 micromol/L, linearity was observed up to 754 micromol/L, with a working concentration range of 0.38-224 micromol/L, inter- and intra-assay coefficients of variation were <6% and <4%, respectively. Samples were stable throughout five freeze-thaw cycles and recovery was between 94% and 114%. CONCLUSIONS: The assay was successfully validated in accordance with FDA guidelines and is suitable for quantitation of choline in research and clinical settings.

A retrospective study of surgically treated cases of septic peritonitis in the cat (2000-2007).

OBJECTIVES: To review aetiology, clinical signs and outcome of cats surgically treated for septic peritonitis (2000-2007). METHODS: A retrospective study. Inclusion criteria were the identification of intracellular bacteria and degenerate neutrophils and/or a positive culture from abdominal fluid and exploratory coeliotomy. Aetiology, clinical signs, haematological and biochemical parameters, surgical treatment and outcome were recorded and analysed. RESULTS: Twenty-six cats fulfilled the inclusion criteria. Abdominal pain was reported in 10 (38 per cent) and vomiting was reported in 11 (42 per cent) of the cats. The most common aetiology was trauma (31 per cent). The principal source of contamination was the gastrointestinal tract. Hyperlactataemia, hypoproteinaemia and hyperglycaemia were reported in 9, 13 and 14 of the 26 cases, respectively. Non-survivors had significantly higher blood lactate concentrations than survivors (P=0.02). Nineteen cats were managed with primary closure, two with closed suction drains and three with open peritoneal drainage. Twelve (46 per cent) cats survived to discharge. CLINICAL SIGNIFICANCE: In cats, lethargy, depression and anorexia were more common clinical signs than abdominal pain. Lactate level at the time of diagnosis may be a useful prognostic indicator in cats. The proportion of cats that survived was lower than previously reported and owners should be given a guarded prognosis.

Simultaneous determination of androstenedione and testosterone in human serum by liquid chromatography-tandem mass spectrometry.

BACKGROUND: We aimed to develop a sensitive assay to quantitate serum concentrations of both androstenedione and testosterone within the female range simultaneously, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in the routine clinical laboratory and to compare this method with immunoassay. METHOD: Samples (200 microL) were prepared by liquid-liquid extraction using (1 mL) methyl-tert-butyl-ether. Deuterated androstenedione and testosterone were used as internal standards. RESULTS: The standard curve was linear to 50 nmol/L, the lower limit of quantitation was 0.25 nmol/L, and intra- and inter-assay coefficients of variation were < 10% for both androgens over the range 0.3-35 nmol/L. There was a poor relationship between the LC-MS/MS and the radioimmunoassay methods for androstenedione with the LC-MS/MS generally giving lower results. For testosterone, the LC-MS/MS and immunoassay methods compared well at all concentrations. However, when female samples only were examined, the agreement deteriorated. CONCLUSIONS: We have developed a sensitive and precise LC-MS/MS method, which gives more accurate results for all androstenedione measurements and low testosterone concentrations than immunoassay.

Development of a method for the measurement of dehydroepiandrosterone sulphate by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Dehydroepiandrosterone sulphate (DHEAS) is a steroid that is increasingly being recognized as a potential drug of abuse in many countries. This is due to its reputation as a hormone that may be able to retard the ageing process. The measurement of DHEAS is useful in the diagnosis of medical conditions such as congenital adrenal hyperplasia and polycystic ovary syndrome. Thus, a liquid chromatography-tandem mass spectrometry method has been developed to determine DHEAS concentrations in human serum. METHOD: The chromatography was performed using a Waters 2795 Alliance HT LC system coupled to a Mercury Fusion-RP column fitted with a SecurityGuard column. RESULTS: DHEAS and the internal standard, deuterated DHEAS, both had a retention time of 1.5 min. The transition determined by the Micromass Quattro tandem mass spectrometer for DHEAS was m/z 367.3>4 96.7 and for the internal standard m/z 369.3>96.6. The method was linear up to 20 micromol/L; the lower limit of detection and the lower limit of quantitation were both 1 micromol/L. The intra- and interassay imprecision were <11% over a concentration range of 1-18 micromol/L for the in-house quality control and <12% for the intra- and interassay imprecision for the Bio-Rad Lyphocheck QC. CONCLUSION: The measurement of DHEAS by liquid chromatography-tandem mass spectrometry is robust and has a simple sample preparation procedure with a rapid cycle time of only 4 min.

Prednisolone measurement in human serum using liquid chromatography tandem mass spectrometry.

Prednisolone is a commonly prescribed corticosteroid used in the treatment of many diseases. Despite high doses of prednisolone, some patients appear to have subtherapeutic concentrations of the drug. It would be useful to measure prednisolone in this group to determine if they have poor absorption or compliance. Hence, we have developed a liquid chromatography-tandem mass spectrometry method for the determination of prednisolone in serum. Chromatography was performed using a C18 column, giving a retention time for both prednisolone and deuterated prednisolone (internal standard) of 1.6 min. Two transitions were monitored for both prednisolone and deuterated prednisolone. These were m/z 361.2 > 343.0 and m/z 361.2 > 146.9 for prednisolone, and m/z 367.2 > 349.0 and m/z 367.2 > 149.9 for the internal standard. The intra- and inter-batch imprecision was < 7% in both cases over a concentration range of 62.5-750 microg/L. The imprecision at the lower limit was 8%, the lower limit of quantitation was determined to be 30 microg/L and the method was linear up to 5000 microg/L. The method allows rapid prednisolone analysis because of a simplified sample extraction step, and has a cycle time of 3.5 min.

Culture counts: why current treatment models fail Hispanic women with type 2 diabetes.

PURPOSE: The purpose of this article is to evaluate whether current treatment models adequately address the cultural factors involved in treatment adherence in Hispanic females with Type 2 diabetes. METHODS: A review of relevant professional literature was conducted. RESULTS: Established health behavior models do not adequately address the unique needs of the female Hispanic population, especially those older women who hold traditional religious and cultural beliefs. CONCLUSIONS: To decrease the devastating effects of Type 2 diabetes among Hispanic women, interventions must be based on a comprehensive, culturally sensitive model that works with cultural values, not against them.

Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene.

The bovine herpesvirus-1 (BHV-1) genome was analysed by Southern blot hybridization using the herpes simplex virus type 1 (HSV-1) DNA polymerase gene as a probe. A 2.5 kilobase region which hybridized specifically to the HSV-1 DNA polymerase gene was identified within the Hind III G fragment at approximate map units 0.334-0.352. In order to provide further evidence that this is the location of the BHV-1 DNA polymerase gene, the 2.5 kilobase region was cloned and part of it sequenced. An uninterrupted stretch of over 800 nucleotides was obtained and an open reading frame spanning the entire sequence was identified. The amino acid sequence that it encodes shows striking homology to a region within the C-terminal half of other herpesvirus DNA polymerases.