Hormone determinations
Most hormones are present in the blood in very low concentrations. This requires special methods that are very complex and prone to failure.
Even before a blood sample is taken it is important to consider the diurnal rhythm, pulsatility and interference (e.g. use of the contraceptive pill). The rapid breakdown of some hormones in the blood necessitates – depending on the hormone being examined – quick processing and targeted preparation of the samples.
Another important feature of hormone determination is the hormone-binding proteins! Only the “free hormones” – that is, only the hormones that are not bound to proteins – are available for the effect! This requires special knowledge to select the right methods for determining the effective hormone concentration – a problem that is far from being solved for all hormones!
The chapter Error Analysis will address the issues of accuracy and precision. In particular, it should be made clear why the values obtained with the same methodology must fluctuate greatly, especially at high and low concentrations!
In the interpretation of the results, age, gender, etc. must also be taken into account.
Development and methods
Almost all of the hormones produced by endocrine glands were discovered by endocrinologists. The method of determining the concentration of messenger substances in the blood was almost always first described in endocrinology laboratories.
The RIA (radioimmunoassay) was developed as an essential tool for the routine determination of hormones, in which antibodies specially cultivated against the corresponding hormone (usually in rabbits) are used with hormones labeled with radioisotopes. The RIA is based on the law of mass action with the associated, unavoidable, large variances in the range of high or low hormone concentrations.
In the IRMA – the Immunoradiometric Assay – two antibodies directed against the hormone are used simultaneously, one firmly bound in the tube, the other radioactively labeled and in dissolved form. As the hormone concentration in the incubate increases, more hormone is bound to the solid antibody. However, the hormone also binds the labeled antibody, so that the radioactivity increases with the concentration of the substance to be measured after decanting the tube.
In addition to the use of radioactive substances, the same examination principles are applied only using other endpoint determinations (EIA, FIA, LIA, ILMA)
Preanalytics
Daily rhythm
Many hormones have a diurnal rhythm. The classic example is the sleep hormone, melatonin. Its concentration at night between 1:00 and 3:00 is around 5 times higher than during the day.
ACTH and cortisolalso show a very significant diurnal rhythm with the highest peaks early in the morning. For this reason, blood should be taken between 8:00 and 9:00 if possible!
In the case of testosterone, in addition to pulsatility, there are also considerable diurnal fluctuations with the highest values in the morning.
Female cycle
The female cycle is triggered and shaped by the female hormones. Without knowing whether a cycle is present, it makes no sense to determine the female hormones. Determining which cycle phase or which cycle disorder is present requires the corresponding determination of the affected hormones.
Pulsatility
Some hormones are released “pulsatile”. For example, GNRH (LHRH) is released from the hypothalamus every 90 minutes. As a result of this phenomenon, the release of LH and FSH is also pulsatile to the same extent. The sex hormones follow this pattern. However, due to the half-life of testosterone and oestradiol, the effects in the blood are much less noticeable. If GnRH secretion does not occur at the correct frequency, this can lead to cycle disorders and infertility, even hypogonadism.
The importance of pulsatility was clearly demonstrated in the treatment of hypothalamic hypogonadism in women. Only pulsatile administration of GNRH every 90 minutes was able to achieve a regular cycle with ovulation and subsequent conception. If GNRH was administered every 60 or 120 minutes, the desired effect did not occur.
If GnRH or its agonists are administered continuously, the release of LH and FSH from the pituitary gland is even completely suppressed, triggering hypogonadism, which is used, for example, in the treatment of prostate cancer.
Interference
Before taking a blood sample, it is important to consider whether the patient is taking preparations that directly interfere with the hormone determination (cross-reaction during the determination), significantly change the concentration of the binding proteins or influence hormone production.
If the possible interferences are not clarified before the blood sample is taken, this can have very problematic consequences. (Case follows)
Stability after blood collection
The stability of the various hormones after blood collection is extremely variable and strongly dependent on the kinetics. A detailed description of the various hormones is not given here and reference is made to the necessary instructions of the laboratory performing the test. The determination of hormones that have not been sent in accordance with the instructions should be avoided at all costs due to possible misinterpretations!
The problem of “free” hormones
Soon after the method for determining the thyroid hormones thyroxine(T4) and triiodothyronine(T3) was described, it was noticed that pregnant women and women taking oestrogens had high thyroid hormones without showing any signs of hyperthyroidism.
This led to the detection of thyroxine-binding globulin(TBG), thyroxine-binding prealbumin(TBPA) and the binding of thyroid hormones to albumin. The concentration of TBG in particular is highly dependent on the concentration of oestrogen. During pregnancy and when taking ovulation inhibitors, TBG concentrations are found that are twice as high as the upper normal value.
With the help of highly complicated dialysis methods, it was possible to determine the formation ratios and thus also the free thyroxine (FT4), which even under normal conditions – i.e. without pregnancy and oestrogen therapy – is only around 0.3 parts per thousand of the total T4 concentration. As the affinity of T3 to TBG is around 90 % lower than that of T4, free T3 (FT3) is around 3 per mill of total T3.
If you consider that the effect of thyroid hormones is mediated at the receptor in the cell nucleus, it becomes clear why the concentration of free thyroid hormones is proportional to their effect; the hormones bound to proteins do not even enter the cell.
The gold standard for determining FT4 and FT3 is the dialysis method, although this would be far too time-consuming for routine use. After much turbulence, the “analog tracer method” for determining free thyroid hormones has established itself with all its pitfalls, especially in problematic situations such as intensive care medicine.
In addition to estrogens, phenothiazines and fibrates can also significantly increase the TBG concentration.
Cortisol
IGF1
Error analysis
The limits of “correctness”