I happened to be looking for some information on the web and came across this regarding the various methods for measuring total and free testosterone in a lab.
Types of testosterone tests
Most of the testosterone in the body binds to carrier proteins (proteins which transport other substances such as hormones through the blood). These carrier proteins are called sex hormone binding globulin (SHBG) and albumin.
Some 44% of testosterone in the blood binds tightly to SHBG and this proportion of testosterone cannot bind to cells in the body which use testosterone (e.g. cells in the testes) because it is bound tightly to and cannot separate from SHBG. This proportion of testosterone is also known as unavailable testosterone, as it is unavailable to the body's cells. A further 54% of testosterone in the blood binds loosely to albumin, but remains available to the body's cells, because it can separate from the albumin and bind to cells which use testosterone. This portion of testosterone is also called bio-available testosterone. The remaining 2% of testosterone in the blood remains unbound and is also available to testosterone-using cells in the body. It is referred to as free testosterone.
Testosterone levels can be measured in terms of total, bio-available or free testosterone and there are various tests which can be used to measure levels of each type of testosterone.
Total testosterone
Total testosterone measures free, albumin bound and SHBG bound testosterone in the blood and is the most commonly used measure of testosterone levels. It is the test usually used to assess testosterone levels in men with suspected hypogonadism or testosterone deficiency. There are several methods available to measure total testosterone.
Liquid or gas chromatography-tandem mass spectrometry
Liquid or gas chromatography, tandem mass spectrometry is the most accurate test or gold standard for measuring total testosterone. Unlike most testosterone tests, it is accurate enough to measure testosterone concentrations in women who have relatively low levels compared to men. However, the test is very complex to perform and although some laboratories in Australia do the test, it is not often used by doctors wishing to assess testosterone levels as it is too costly and time consuming.
Radioimmunoassay test kit
Radioimmunoassay test kits provide a relatively simple means of assessing testosterone levels. However, they are less accurate than liquid or gas chromatography-tandem mass spectrometry and unsuitable for assessing testosterone levels in women.
The test kits contain reagents which are added to an individual's blood sample. Testosterone antibodies with radioactive particles in the reagent bind to testosterone in the sample and the testosterone level is determined by counting the number of testosterone molecules bound to testosterone antibodies. They can be easily counted because of the radioactive particles to which they are attached.
Automated immunoassay systems
Automated platform immunoassay systems are testosterone tests in which most steps (e.g. adding the blood sample, adding reagents) are performed automatically by a machine, rather by people in the laboratory. These tests are the fastest method of assessing testosterone levels. They are considered accurate enough for assessing testosterone levels in men but not in women.
Bio-available testosterone
Bio-available testosterone is a measure of both free and albumin-bound testosterone, both of which are bio-available, or available for use by cells in the body.
Ammonium sulphate precipitation
The ammonium sulphate precipitation method is a test which measures bio-available testosterone. To perform this test, a chemical called aluminium sulphate is added to a blood sample. This causes SHBG-bound testosterone to precipitate (separate out and form a solid substance) from the sample. Bio-available testosterone (free and albumin-bound) remains in the sample. The total testosterone level of the sample (after precipitation of the SHBG-bound testosterone) is then measured. Aluminium sulphate precipitation is considered the gold standard (most accurate) measure of bio-available testosterone. It is however impractical for routine use by doctors, as it is too time-consuming and costly to perform.
Calculated bio-available testosterone
It is also possible to calculate bio-available testosterone using an equation, if total testosterone, SHBG and albumin levels are known.
Free testosterone
A test for free testosterone measures only the 2% of testosterone which remains unbound to proteins in blood. Tests are also available to measure free testosterone in saliva, however these are relatively new, require further testing and are currently only recommended for use in research studies and not for use by doctors for diagnosing patients. Free testosterone levels may be measured in men whose total testosterone test results showed testosterone levels which are on the borderline of levels considered to indicate testosterone deficiency. Free testosterone levels are often assessed in women with suspected testosterone abnormalities. Free testosterone can be measured by a number of methods.
Equilibrium dialysis
Equilibrium dialysis is a complex measure of free testosterone in which a blood sample is diluted and passed through a special semi-permeable membrane into a buffer solution. Only some of the testosterone in the sample can pass through the membrane- the rest remains in the blood sample. After filtering the sample through the semi-permeable membrane, the amount of testosterone in the buffer solution and the sample is measured and used to calculate the concentration of free testosterone This test is considered the gold standard for measuring free testosterone but is impractical for routine use.
Centrifugal ultrafiltration
Centrifugal ultrafiltration is also an accurate measure of free testosterone, however considered impractical for routine use. The method involves adding a substance called testosterone tracer (usually a coloured substance which binds to testosterone and can then be easily identified because of its colour) to blood samples, which are then incubated at 37oC and centrifuged (spun at a high speed) to separate the testosterone in the sample. The concentration of free testosterone can then be measured by examination of the filtrate (the separated portion of the sample).
Direct immunoassay test kits
Direct immunoassay test kits are the easiest and fastest available methods of assessing free testosterone. They are test kits in which reagents containing testosterone antibodies and tracer particles (either radioactive or non-radioactive) which bind to free testosterone in the sample. The tracer particles are easily identifiable and can be counted to measure the concentration of free testosterone in the sample. The results are less accurate than other methods though and lower results are typically obtained compared to equilibrium dialysis.
Calculated free testosterone
There are also a number of equations for calculating the concentration of free testosterone in a blood sample, without making an actual measurement of free testosterone. Other substances in the blood which interact with testosterone are measured instead. For example, measuring total testosterone and sex hormone binding globulin allows a measure of total testosterone called the Free Androgen Index to be calculated.
