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Microsoft word - hormone analyses at the izw
Hormone analyses at the IZW
The endocrine laboratory of the Leibniz Institute for Zoo and Wildlife Research
(IZW) offers measurement of steroid hormones in blood plasma, urine and faecal samples (non-invasive monitoring of hormones) on a service basis. We are able to analyse progesterone, estradiol, testosterone, cortisol, corticosterone, prostaglandin F2α-metabolite (PGFM) in blood, and their metabolites in urine and faeces reflecting male and female reproductive and adrenocortical activity (stress).
1. Requirements regarding sample collection and samples
1.1 Blood samples
To analyse progesterone, testosterone, cortisol, and corticosterone, we require a minimum amount of 50 µl plasma. For the analysis of estradiol we need 0.2 ml plasma due to the low estradiol concentrations (in the pg range).
1.2 Urine and faecal samples
For measurements in urine we require 1 ml of urine. For the analysis of faecal samples we use 0.5 g. Sample amounts may be reduced for small mammals or birds. All samples have to be kept frozen (-20°C) immediately after collection, otherwise changes in metabolite concentrations may occur due to microbial activities, distorting or invalidating the results.
Please keep in mind that the secretion of cortisol / corticosterone follows diurnal and pulsatile patterns. Therefore, it is impossible to measure adrenal activity (stress) in an animal based on a single sample. Therefore, samples should be collected over a longer time period.
The same applies for reproductive monitoring. A single elevated progesterone concentration in samples of plasma, urine or faeces does not necessarily indicate pregnancy, it may just as well indicate the luteal phase of a cycling female. Thus a sample collection period exceeding the length of an ovarian cycle is necessary. Pregnancy can only be confirmed based on persisting high progesterone concentrations in samples collected over a certain period.
Each animal species metabolizes steroids in a unique manner, and so-called biological validation
of a non-invasive hormone measurement must be performed prior to monitoring reproductive activity in a valuable animal. Biological validation tests the ability of a certain protocol to detect known differences or changes in hormone levels.
For this reason, we can offer non-invasive hormone monitoring with guaranteed results only for species for which we have prior experience or ready-to-use analytical methods.
Please note that if the results are to be published in a scientific journal, referees will most likely require assay evaluation and biological validation.
Blood sample (direct measurement): 6.00 € Blood sample (measurement with prior extraction): 8.00 € Urine samples (including hydrolysis and extraction): 8.00 € Faecal samples (including extraction): 11.00 €
4. Customer risks
If a customer explicitly requests non-invasive hormone measurement for a species for which we have no analytical expertise, we cannot guarantee reliable results. To minimize this risk, we ask the customer to provide us with additional reference samples for biological validation.
Reference samples e.g. for faecal testosterone metabolite analyses from subadult males should reveal distinct lower concentrations than samples from adult males. If this is not the case, we consider the biological validation failed and immediately stop the analyses.
The costs listed above will have to be paid for all procedures we conducted, irrespective of whether the analyses are successful.
Delivery and handling time of samples will be estimated upon consultation.
A special case is parturition monitoring in elephants where analyses have to be carried out 3 times per week. When hormone concentrations drop, we switch to daily measurements also including weekends.
Raw data (reader print-outs) are saved in folders in the room of the lab leader. In addition, all EIA protocols are scanned and saved as pdf files with a name following the template: EIA-5aP-20120219, where 5a-P defines the EIA. Data evaluation is carried out with Excel
and the calculated hormone concentrations are saved in Excel
sheets. From each sheet a link is given to the corresponding EIA (e.g. EIA-5aP-20120219) to connect the calculated concentrations to the corresponding raw data.
6. Quality management
We use analytical methods that in most cases have been published in peer-reviewed journals including quality criteria and thus have passed critical assessment of external experts. In addition, each assay is routinely subjected to the following quality management procedures:
1. Sample analyses are carried out in duplicates. In case duplicates deviate more than
2. Standard samples (controls) are included in each assay. These must always result in
the same concentrations, irrespective of the person running the assay. Results are documented over several years in a central data set and are under control of the lab leader. In case of significant deviations from normal, the assay has is repeated. In case of repeated deviations a troubleshooting routine is initiated.
3. Calibrations curves are used for quantification. The calibration curves are compared
according to their B10, B20, B50 and B80 values (B50 is the hormone concentration that is needed to reduce absorption and thus binding of the label by 50%). B-values are calculated by the Magellan software (Tecan) and must remain constant.
Regarding non-invasive monitoring of hormones ring tests are not feasible because every laboratory uses its own unique assays based on a distinct antibody. For non-invasive hormone monitoring, biological validation is the most important prerequisite for generating meaningful conclusions.
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