Blood hormone and tumor marker concentrations are usually determined by immunochemical methods which are based on an unique reaction between antigen and assay capture antibody. Despite the speed and simplicity of assays performance on automatic immunochemistry platforms, the interpretation of final results requires a deep knowledge of method fallibility. General lack of immunoassays standardization, presence of cross-reacting substances in patient’s sample, limitation of free hormones measurement due to abnormal analyte binding protein concentrations, assay interferences due to patient’s autoantibodies, and heterophilic antibodies, as well as proper interpretation of very low- and very high-sample analyte levels, are the main points discussed in respect to hormones and tumor markers measurement in geriatric population.
Aging process is associated with physiological changes in function of almost every organ and system, including the endocrine system. The function of endocrine glands function declines progressively with age. For example, dehydroepiandrosterone sulphate (DHEAS) concentration is about 10–20% of maximum in patients at the age of 70–80 years [
Hormones, proteins, peptides, tumor markers, and drugs are routinely measured using automated immunochemistry platforms. Immunochemistry methods are based on the reaction between an antigen and an antibody; both competitive and noncompetitive method formats are used. The reaction between antigen and antibody is very specific due to unique properties and stereochemistry of epitope on the antigen and paratope on the antibody. Although the analytical procedure for measuring hormones is very simple and easy to perform, the interpretation of results requires not only medical knowledge but also deep knowledge of immunochemistry limitations. This is especially true when hormone measurements are performed in the serum of the patient at an extremely advanced age, the patient with multiple or chronic disease, and the patient on multiple drugs therapy.
For all the laboratory determinations, the preanalytical phase of a diagnostic procedure contributes the most to the total laboratory error of measurement, regardless of the patient’s age. The type of anticoagulants used, the presence of hemolysis, lipemia, hyperbilirubinemia, and paraproteinemia are well-known factors, influencing the measurement of biochemical markers, including hormones. The observed bias due to hemolysis may be negative, as it is the case with cortisol, parathyroid hormone (PTH), and insulin measurement, or positive as it is the case with troponin I determination [
Apart from the aforementioned pre non-specific analytical problems, there are many pitfalls which may occur during the analytical phase of hormones determination by immunochemical methods, which are known by the laboratory personnel but frequently unknown by physicians. For a proper interpretation of the hormone concentration results, the comparison of the results with appropriate reference intervals coupled with good clinical knowledge is necessary. In case of discrepancy between the laboratory data and the clinical picture of the patient, repeated analytical measurements are usually requested. However, in the case of hormones and tumor markers, repeated measurements of the analyte by immunochemistry in questionable patients’ samples give concentration results that do not always meet clinical expectations. To avoid such a situation, it is important for clinicians to know and to understand the limitation and fallibility of immunochemical methods in order to protect the patient from misdiagnosis. This is extremely important for every patient, but it must be stressed that in samples from geriatric patients, the presence of various drugs and their metabolites, the presence of autoantibodies and other inducible antibodies, and low albumin level, as well as disturbances in specific and nonspecific hormone-binding protein levels, are frequently observed. In addition, tumor marker measurements are much more frequently requested in older patients as compared to other age groups, extremely high level of some proteins can be expected as well. On the other hand, after surgery of the endocrine gland due to cancer, or during suppressive therapy, the measurement of very low level of some hormones is important for clinical management of a geriatric patient. Thus, for proper interpretation of the laboratory results of hormones and tumor markers determination, it is advisable for physicians to become familiar with most important immunochemistry issues, so that they could answer the following questions: (a) what is being measured by a given immunoassay? (b) how accurate are low and high concentrations of hormone/tumor marker measured? (c) how do binding proteins affect hormone measurement? (d) how do autoantibodies; heterophilic and anti-animal antibodies interfere with the measurement of hormone/tumor marker?
Different chemical molecules, such as protein, peptides, biogenic amines, steroids, and drugs, can be measured by immunochemical methods. As for any other methods, standardization of immunochemical methods is necessary to ensure accuracy of a measurement and comparability of results between different assays. However, most of the immunoassays lack proper standardization. Although the primary standards are available for small molecules (amines, steroids, and drugs), the lack of commutability between primary or secondary standards and the patient’s samples due to matrix effect make the standardization process a very difficult task. On the other hand, many hormones of clinical interest are present in the blood in heterogeneous forms (growth hormone, prolactin, gonadotropins, TSH, and gastrin) [
Immunochemical methods for the measurement of the same analyte may differ with respect to reagent antibodies and to a different standard for calibration. As a consequence, the results of the concentration of hormones and tumor markers obtained by different assay or immunochemistry platforms are often not comparable. Thus, two issues are of great importance: firstly, the knowledge of the molecule that is being measured by immunoassay; secondly, the mandatory use of the method-dependent reference intervals established by the laboratory. Taking into account the lack of immunoassays standardization, heterogeneity of many peptides and protein, structural similarities of steroids and their metabolites, as well as capture antibody specificity, the request for the hormone or tumor marker measurement by two laboratories using different immunoassays should be avoided.
Analytical sensitivity is an important issue for those analytes for which low concentrations in the patient’s sample are diagnostically important as it can be observed in geriatric population in case of C-reactive protein (CRP), estrogen, TSH, and troponin measurement [
Measurement of very high or extremely high concentration of hormones and tumor markers is a great challenge for laboratory staff, since disagreement between the clinical picture of the patient and the laboratory result is sometimes noted. This is especially true for a geriatric population because the frequency of oncologic diseases of different origin increases with age. In immunochemical noncompetitive methods, unlike in other analytical methods, a high-dose effect (hook effect) may occur. In such methods, antigen is linked with two assay antibodies (solid-phase capture antibody and signal antibody) forming a so-called “sandwich”, and the proportionality between the assay signal and analyte concentration is seen. However, the enormous amount of the analyte in the patient’s sample blocks both assay capture and labeled antibodies, which does not allow for the formation of a typical “sandwich” [
In the geriatric population, the results of hormones measurement should be interpreted with caution as age-related decline in concentration is characteristic not only for specific binding proteins, such as insulin-like growth factor-binding protein 3 (IGFBP-3), but also for nonspecific binding proteins, such as albumin [
A good example of the effect of binding proteins on hormone determination is the estimation of free-thyroid hormone levels (FT4 and FT3) in elderly population. The FT4 plasma concentration depends on the binding capacity of thyroxine-binding globulin concentration (TBG) as well as albumin and prealbumin. Depending on the immunoassay format, either false positive (competitive methods) or false negative (noncompetitive methods) results of free hormone measurement can be obtained as an effect of binding proteins interference [
In geriatric patients treated with heparin (including low-molecular-weight heparin), a misleading diagnosis can affect the patients’ safety due to falsely elevated FT4. The concentration of FT4 in such patients depends on the time that elapsed between heparin administration and blood sampling as well as the time that elapsed between the collection of the blood and performing immunoassay measurement [
Lower serum albumin level frequently observed in geriatric population is also associated with the decrease in maximum binding capacity of drugs, which is significant during polytherapy. As a consequence, free-drug concentrations in the plasma are increased [
Common health problems encountered in the geriatric population include various chronic inflammatory diseases such as rheumatoid arthritis, pneumonia, and systemic lupus erythematosus (SLE) [
From the analytical point of view, different problems have to be considered if it is necessary to measure the concentration of protein against which autoantibodies are present in the plasma sample or measurement of autoantibodies as an independent marker of immune disease is requested. Firstly, autoantibodies can interfere with the analyte measurement, giving erroneous results and, depending on the assay format, both underestimation (noncompetitive methods) or overestimation (competitive methods) can be observed [
In addition to autoantibodies against self-antigens, any inducible antibodies against different foreign antigens can be present in human serum samples. Such inducible antibodies are usually polyreactive and are directed against poorly defined foreign antigens. For analytical purposes they have been called heterophilic antibodies. The best known heterophilic antibodies are rheumatoid factors [
In the geriatric population, interference from heterophilic antibodies is as extremely important as the variety of antibodies that are present in the blood. For example, rheumatoid factors are present in 70% of patients with rheumatoid arthritis and the occurrence of human anti-mouse antibodies as a consequence of treating the patient with mouse immunoglobulin for diagnostic and therapeutic purposes is estimated as high as 11.7% [
Accuracy of analytical measurement of different biochemical parameters is a prerequisite for proper diagnosis and treatment monitoring of the patient. Immunochemical methods play an important role in measurement of a variety of biochemical molecules, although due to their fallibility, many limitations in measurement are noted. Immunochemistry is a very powerful analytical technique, but imperfections in analytical measurement are directly connected with unique basis of methods, general lack of standardization, and presence of many interfering substances in patients’ samples. The more a patient’s sample matrix differs from the normal sample matrix, the higher the probability that erroneous results will occur. In older patients, misinterpretation of immunochemistry results due to the presence of interfering endogenous substances (cross-reacting substances, abnormal hormone binding proteins, presence of autoantibodies, heterophilic antibodies, and anti-animal antibodies) in the blood is more frequent than in younger individuals. It has to be stressed, that most pitfalls in analyte measurement by immunochemistry are related to a patient’s sample, and no quality control assurance program exists to protect patient from erroneous results. The only way to suspect an error in immunochemistry results is through the information obtained from physicians, where there is disagreement between laboratory results and the patient’s condition. Each laboratory has procedures to look for errors in immunochemistry measurement, but the information must first come from clinicians. The more signals from physicians, the higher the possibility in avoiding immunochemistry errors in the future. In order to achieve this, the physician taking care of the geriatric population should be familiar with the limitations of immunochemistry.
C-reactive protein
Free thyroxine
Insulin-like growth factor
Parathyroid hormone
Rheumatoid factors
Thyroxine-binding globulin
Thyroid-stimulating hormone.
The authors declare that they have no conflict of interests.