madman
Super Moderator
* The Centers for Disease Control and Prevention (CDC) has established a program to standardize and improve the measurement of steroid hormones, including E2 and T, using liquid chromatography-tandem MS (LC-MS/MS). In addition, the CDC has partnered to establish postmenopausal reference ranges for T and is developing reference intervals for E2. Minimizing some of the technical limitations of the current immunoassays and MS assays should provide better and more accurate assays for patient care.
* Equilibrium dialysis combined with a MS assay is widely considered as the “gold standard” for determining the concentration of free T.36
* Immunoassays to measure free T directly in serum and plasma are available but are not recommended,37mainly because of their high inaccuracy compared with equilibrium dialysis methods or calculation procedures.38,39 Studies have shown that this assay method significantly underestimates free T concentrations compared with corresponding values obtained by equilibrium dialysis.36,40,41
* Since MS assays commonly differ by their use of MS and chromatographic equipment, operational conditions, and extraction procedures, the CDC has standardized E2 and T MS assays as well as immunoassays. This is essential to obtain accuracy and uniformity of test results among different laboratories. In addition, the CDC HoSt program provides valuable technical assistance to participants who analyze E2 and/or T in serum samples obtained from the CDC. Also of importance is the establishment of postmenopausal reference ranges for E2 and T by the CDC. These reference ranges are scarce but are essential for providing accurate test results.
Abstract
Circulating concentrations of estradiol (E2) and testosterone (T) have been measured in postmenopausal women for many years, predominantly by immunoassays and mass spectrometry (MS) assays. Although the accuracy of E2 and T assays is very important to provide clinically meaningful results, there are challenges in establishing their accuracy. MS assays demonstrate higher accuracy for steroid hormone measurements, but immunoassays can provide clinically meaningful results, especiallyat high concentrations. The Centers for Disease Control and Prevention (CDC) has established a program to standardize and improve the measurement of steroid hormones, including E2 and T, using liquid chromatography-tandem MS (LC-MS/MS). In addition, the CDC has partnered to establish postmenopausal reference ranges for T and is developing reference intervals for E2. Minimizing some of the technical limitations of the current immunoassays and MS assays should provide better and more accurate assays for patient care.
The purpose of the present manuscript is (1) to point out the challenges involved in developing accurate assays for measurement of E2 and T in postmenopausal women, (2) to discuss the importance of standardizing steroid hormone assays, and (3) to emphasize the need for clinicians to understand the differences, as well as the advantages and disadvantages, of immunoassays and MS assays.
IMMUNOASSAYS
The elimination of purification steps and the use of low sample volumes contribute largely to the limitations observed with today’s automated chemiluminescent immunoassays. With the purification steps no longer performed, more interfering compounds are in the sample, leading to overestimated E2 measurements. This is especially evident when E2 is measured in serum from postmenopausal women treated with an exogenous estrogen,where overestimation can be > 10 times higher.15 Furthermore, direct immunoassays lack the sensitivity to measure reliably baseline postmenopausal serum E2 concentrations. These assays are not able to measure low E2 concentrations (generally < 25 pg/mL) accurately. As reported in one study, the reportable range for most assays does not reach below 10 pg/mL and those assays reaching below 10 pg/mL show a high positive measurement bias at these low E2 concentrations.16 E2 levels in patients responding to aromatase inhibitor therapy typically have E2 levels below 10 pg/mL. Most direct assays would not be sufficiently sensitive to monitor patients on aromatase inhibitor therapy or may falsely indicate that the goal of such a therapy has not been reached, leading to an inappropriate change in therapy.17
Furthermore, the accuracy and reliability of direct E2 assays are subject to the same limitations as other immunoassays, such as those related to biotin interference and the presence of heterophilic antibodies. Exogenous biotin has been recognized as an interference factor in streptavidin-biotin‐based immunoassays because biotin in serum samples competes with biotinylated reagents for binding sites in streptavidin, causing false increases in measurement values in competitive hormone assays. Although typical dietary intake of biotin appears to be insufficient to affect streptavidin-biotin based assays, supplements containing 10 mg or more of biotin can causes purious results.18 The presence of heterophilic antibodies,though rare, can also lead to falsely elevated E2 measurements.19
Some assays may not fully capture the amount of E2 or T present. Since a substantial amount of E2 and T is bound tightly to sex hormone binding globulin (SHBG), it is essential to dissociate these steroids from SHBG, otherwise their levels will be underestimated. This is usually done by addiing a reagent with a low pH in the assay. Thus, the results of these assays will depend on theeffectiveness of displacement from SHBG.
MASS SPECTROMETRY ASSAYS
A major strength of MS assays is their potential to achieve high analytical specificity and sensitivity, resulting in accurate and precise measurement. Another strength,compared with immunoassays, is their ability to multiplex compounds in a relatively small volume of serum, plasma,or urine. However, MS assays have certain limitations. Accurate and precise quantitation requires the use of internal standards to account for procedural losses. The development and availability of appropriate internal standards is not easy to achieve, especially for complex metabolites. In addition, this high level of analytical performance can only be achieved through validation by the laboratory operating the assay. Furthermore, while the MS assays use the same technical principles, they commonly differ by their use of MS and chromatographic equipment and operational conditions, and employ different extraction procedures. However, MS assays are often erroneously perceived as a homogenous group of assays with very similar performance characteristics. This is, for example, reflected in MS assays being listed as one peer group in proficiency testing/external quality assessment (PT/EQA) programs, while immunoassays are separated into multiple peer groups. Peer group evaluation isused to determine whether a laboratory is using a measurement procedure that conforms to a manufacturer’s specifications and/or to other laboratories using the same technology.
MS assays are still technically demanding and complex, and they can be expensive to operate. Furthermore, they require a highly trained technician to operate the instrument. Finally, the overall costs of implementing and using MS assays to measure steroid hormones prohibit their use in small laboratories. However, the newest developments of fully automated and integrated HPLC-MS/MS systems for clinical use seem to overcome some of these limitations.24,25
STUDIES COMPARING ESTRADIOL AND TESTOSTERONE MEASUREMENTS BY IMMUNOASSAY AND MASS SPECTROMETRY
* Estradiol assays
* Testosterone assay
Free testosterone
In clinical practice, two forms of T are measured: total and free (unbound). In the circulation, T is bound to SHBG and albumin, and a small fraction (1%-2%) is free; this comprises total T. Free T is important because it is this fraction that binds to the androgen receptor and exhibits biological activity. Monitoring free T concentrations can help identify potential androgen imbalance in conditions such as PCOS. The 2023 international PCOS guidelines recommend use of both total and free T as the first line laboratory tests to assess biochemical hyperandrogenism in the diagnosis of PCOS.35
Equilibrium dialysis combined with a MS assay is widely considered as the “gold standard” for determining the concentration of free T.36 The equilibrium dialysis procedure determines the percentage of free T in serum or plasma, and this percentage is then multiplied by the total T concentration, which should be measured by an accurate assay. The procedure involves dialysis of serum or plasma samples in a small buffer-filled container with a semipermeable cellulose membrane that has a low molecular weight cutoff. After adding the sample to the container, the protein (SHBG and albumin)-bound T is too large to cross the membrane, but free T can readily cross the membrane and be measured. While measurement of free T in serum from male patients is typically easy toaccomplish by LC-MS/MS, this measurement can be challenging in sera from female patients. For those situations, an indirect method is often used. This method requires a radioactive tracer (3H-T) to be added to the sample before dialysis. The proportion of tracer in the dialysate provides a measure of the percentage of free T,which is then multiplied by the total T concentration measured by LC-MS/MS.
Immunoassays to measure free T directly in serum and plasma are available but are not recommended,37mainly because of their high inaccuracy compared with equilibrium dialysis methods or calculation procedures.38,39 Studies have shown that this assay method significantly underestimates free T concentrations compared with corresponding values obtained by equilibrium dialysis.36,40,41
Although the equilibrium dialysis assay can accurately measure free T, the method is subject to sources of error that may contribute to inaccuracy and imprecision.36 Furthermore, due to the operational complexities in carrying out the equilibrium dialysis assay and difficulties in automating the assay, only a limited number of laboratories perform the assay.
Because of these limitations, an accurate algorithm,validated against the equilibrium dialysis assay, was developed to provide calculated free T concentrations.36 The algorithm is based on a linear model of T binding to SHBG and albumin, and requires measurement of total T ,SHBG, and albumin. Although systematic differences have been reported in some studies that compared the calculated free T method with the equilibrium dialysisassay, it has been and continues to be widely used in many laboratories.36,42,43
Another method that is often used to determine freeT is the free androgen index, which is calculated from the ratio of total T/SHBG (in molar units). Although this method is simplistic, it is considered to be an inaccurate measure of free T concentrations, and most experts do not favor its use.3
Some laboratories offer salivary T measurements as an indicator for free T. There is a misconception that sex steroid hormones measured in saliva represent the free fraction of the steroid. The presence of steroid-metabolizing enzymes in salivary glands has been reported in some studies.44 For example, androstenedione was isolated in an in vitro study in which 14C-labeled T was incubated with human parotid and submandibular salivary glands from women and men.45 Thus, salivary T is not considered to be an accurate marker of circulating free T concentrations.
* Standardization of testosterone and estradiol assays
* Reference ranges of estradiol and testosterone
CONCLUSIONS
Analytical accuracy of hormone assays is critical because inaccurate assays can lead to misdiagnosis or an incorrect assessment of treatment. For many years, E2 and T have been measured predominantly by immunoassays and MS assays, which have very different methodological principles. In recent years, E2 and T have been quantified by direct chemiluminescent immunoassays on automated platforms and by LC-MS/MS assays. Although these assays are used widely for clinical diagnostic testing and in research studies, establishing optimal measurements of these hormones is hampered bytechnical limitations of the assay methods. This is especially true of the chemiluminescent immunoassays, which lack sensitivity for quantifying low serum E2 concentrations in women and specificity for quantifying E2 concentrations following treatment with exogenous estrogens in postmenopausal women. In addition, studiesshow that direct immunoassays should not be used for the measurement of T in women. Without a purificationstep before quantitation of E2 or T by immunoassay, these hormones will not be measured accurately due tocross-reaction of some of their metabolites with the antibody in the assay. Despite the deficiencies in chemiluminescent immunoassays, these assays continue to provide clinically meaningful results, especially at higher concentrations, eg, in assisted reproductive technology protocols where E2 concentrations are elevated. In contrast to immunoassays, a major strength of MS assays is their potential to achieve high analytical specificity and sensitivity, resulting in accurate and precise measurements of E2 and T. In addition, MS assays allow ahigh degree of multiplexing for measuring multiple metabolites in a single run using a relatively small amount of serum or urine. However, MS assays have certain limitations. Accurate and precise measurements requir ethe use of internal standards to monitor procedural losses, which are difficult to prepare. In addition, MS assays are complex, technically demanding, expensive,and require a highly trained technician to operate the instrument
Since MS assays commonly differ by their use of MS and chromatographic equipment, operational conditions,and extraction procedures, the CDC has standardized E2 and T MS assays as well as immunoassays. This is essential to obtain accuracy and uniformity of test results among different laboratories. In addition, the CDC HoSt program provides valuable technical assistance to participants who analyze E2 and/or T in serum samples obtained from the CDC. Also of importance is the establishment of postmenopausal reference ranges for E2 and T by the CDC. These reference ranges are scarce but are essential for providing accurate test results.
* Equilibrium dialysis combined with a MS assay is widely considered as the “gold standard” for determining the concentration of free T.36
* Immunoassays to measure free T directly in serum and plasma are available but are not recommended,37mainly because of their high inaccuracy compared with equilibrium dialysis methods or calculation procedures.38,39 Studies have shown that this assay method significantly underestimates free T concentrations compared with corresponding values obtained by equilibrium dialysis.36,40,41
* Since MS assays commonly differ by their use of MS and chromatographic equipment, operational conditions, and extraction procedures, the CDC has standardized E2 and T MS assays as well as immunoassays. This is essential to obtain accuracy and uniformity of test results among different laboratories. In addition, the CDC HoSt program provides valuable technical assistance to participants who analyze E2 and/or T in serum samples obtained from the CDC. Also of importance is the establishment of postmenopausal reference ranges for E2 and T by the CDC. These reference ranges are scarce but are essential for providing accurate test results.
Abstract
Circulating concentrations of estradiol (E2) and testosterone (T) have been measured in postmenopausal women for many years, predominantly by immunoassays and mass spectrometry (MS) assays. Although the accuracy of E2 and T assays is very important to provide clinically meaningful results, there are challenges in establishing their accuracy. MS assays demonstrate higher accuracy for steroid hormone measurements, but immunoassays can provide clinically meaningful results, especiallyat high concentrations. The Centers for Disease Control and Prevention (CDC) has established a program to standardize and improve the measurement of steroid hormones, including E2 and T, using liquid chromatography-tandem MS (LC-MS/MS). In addition, the CDC has partnered to establish postmenopausal reference ranges for T and is developing reference intervals for E2. Minimizing some of the technical limitations of the current immunoassays and MS assays should provide better and more accurate assays for patient care.
The purpose of the present manuscript is (1) to point out the challenges involved in developing accurate assays for measurement of E2 and T in postmenopausal women, (2) to discuss the importance of standardizing steroid hormone assays, and (3) to emphasize the need for clinicians to understand the differences, as well as the advantages and disadvantages, of immunoassays and MS assays.
IMMUNOASSAYS
The elimination of purification steps and the use of low sample volumes contribute largely to the limitations observed with today’s automated chemiluminescent immunoassays. With the purification steps no longer performed, more interfering compounds are in the sample, leading to overestimated E2 measurements. This is especially evident when E2 is measured in serum from postmenopausal women treated with an exogenous estrogen,where overestimation can be > 10 times higher.15 Furthermore, direct immunoassays lack the sensitivity to measure reliably baseline postmenopausal serum E2 concentrations. These assays are not able to measure low E2 concentrations (generally < 25 pg/mL) accurately. As reported in one study, the reportable range for most assays does not reach below 10 pg/mL and those assays reaching below 10 pg/mL show a high positive measurement bias at these low E2 concentrations.16 E2 levels in patients responding to aromatase inhibitor therapy typically have E2 levels below 10 pg/mL. Most direct assays would not be sufficiently sensitive to monitor patients on aromatase inhibitor therapy or may falsely indicate that the goal of such a therapy has not been reached, leading to an inappropriate change in therapy.17
Furthermore, the accuracy and reliability of direct E2 assays are subject to the same limitations as other immunoassays, such as those related to biotin interference and the presence of heterophilic antibodies. Exogenous biotin has been recognized as an interference factor in streptavidin-biotin‐based immunoassays because biotin in serum samples competes with biotinylated reagents for binding sites in streptavidin, causing false increases in measurement values in competitive hormone assays. Although typical dietary intake of biotin appears to be insufficient to affect streptavidin-biotin based assays, supplements containing 10 mg or more of biotin can causes purious results.18 The presence of heterophilic antibodies,though rare, can also lead to falsely elevated E2 measurements.19
Some assays may not fully capture the amount of E2 or T present. Since a substantial amount of E2 and T is bound tightly to sex hormone binding globulin (SHBG), it is essential to dissociate these steroids from SHBG, otherwise their levels will be underestimated. This is usually done by addiing a reagent with a low pH in the assay. Thus, the results of these assays will depend on theeffectiveness of displacement from SHBG.
MASS SPECTROMETRY ASSAYS
A major strength of MS assays is their potential to achieve high analytical specificity and sensitivity, resulting in accurate and precise measurement. Another strength,compared with immunoassays, is their ability to multiplex compounds in a relatively small volume of serum, plasma,or urine. However, MS assays have certain limitations. Accurate and precise quantitation requires the use of internal standards to account for procedural losses. The development and availability of appropriate internal standards is not easy to achieve, especially for complex metabolites. In addition, this high level of analytical performance can only be achieved through validation by the laboratory operating the assay. Furthermore, while the MS assays use the same technical principles, they commonly differ by their use of MS and chromatographic equipment and operational conditions, and employ different extraction procedures. However, MS assays are often erroneously perceived as a homogenous group of assays with very similar performance characteristics. This is, for example, reflected in MS assays being listed as one peer group in proficiency testing/external quality assessment (PT/EQA) programs, while immunoassays are separated into multiple peer groups. Peer group evaluation isused to determine whether a laboratory is using a measurement procedure that conforms to a manufacturer’s specifications and/or to other laboratories using the same technology.
MS assays are still technically demanding and complex, and they can be expensive to operate. Furthermore, they require a highly trained technician to operate the instrument. Finally, the overall costs of implementing and using MS assays to measure steroid hormones prohibit their use in small laboratories. However, the newest developments of fully automated and integrated HPLC-MS/MS systems for clinical use seem to overcome some of these limitations.24,25
STUDIES COMPARING ESTRADIOL AND TESTOSTERONE MEASUREMENTS BY IMMUNOASSAY AND MASS SPECTROMETRY
* Estradiol assays
* Testosterone assay
Free testosterone
In clinical practice, two forms of T are measured: total and free (unbound). In the circulation, T is bound to SHBG and albumin, and a small fraction (1%-2%) is free; this comprises total T. Free T is important because it is this fraction that binds to the androgen receptor and exhibits biological activity. Monitoring free T concentrations can help identify potential androgen imbalance in conditions such as PCOS. The 2023 international PCOS guidelines recommend use of both total and free T as the first line laboratory tests to assess biochemical hyperandrogenism in the diagnosis of PCOS.35
Equilibrium dialysis combined with a MS assay is widely considered as the “gold standard” for determining the concentration of free T.36 The equilibrium dialysis procedure determines the percentage of free T in serum or plasma, and this percentage is then multiplied by the total T concentration, which should be measured by an accurate assay. The procedure involves dialysis of serum or plasma samples in a small buffer-filled container with a semipermeable cellulose membrane that has a low molecular weight cutoff. After adding the sample to the container, the protein (SHBG and albumin)-bound T is too large to cross the membrane, but free T can readily cross the membrane and be measured. While measurement of free T in serum from male patients is typically easy toaccomplish by LC-MS/MS, this measurement can be challenging in sera from female patients. For those situations, an indirect method is often used. This method requires a radioactive tracer (3H-T) to be added to the sample before dialysis. The proportion of tracer in the dialysate provides a measure of the percentage of free T,which is then multiplied by the total T concentration measured by LC-MS/MS.
Immunoassays to measure free T directly in serum and plasma are available but are not recommended,37mainly because of their high inaccuracy compared with equilibrium dialysis methods or calculation procedures.38,39 Studies have shown that this assay method significantly underestimates free T concentrations compared with corresponding values obtained by equilibrium dialysis.36,40,41
Although the equilibrium dialysis assay can accurately measure free T, the method is subject to sources of error that may contribute to inaccuracy and imprecision.36 Furthermore, due to the operational complexities in carrying out the equilibrium dialysis assay and difficulties in automating the assay, only a limited number of laboratories perform the assay.
Because of these limitations, an accurate algorithm,validated against the equilibrium dialysis assay, was developed to provide calculated free T concentrations.36 The algorithm is based on a linear model of T binding to SHBG and albumin, and requires measurement of total T ,SHBG, and albumin. Although systematic differences have been reported in some studies that compared the calculated free T method with the equilibrium dialysisassay, it has been and continues to be widely used in many laboratories.36,42,43
Another method that is often used to determine freeT is the free androgen index, which is calculated from the ratio of total T/SHBG (in molar units). Although this method is simplistic, it is considered to be an inaccurate measure of free T concentrations, and most experts do not favor its use.3
Some laboratories offer salivary T measurements as an indicator for free T. There is a misconception that sex steroid hormones measured in saliva represent the free fraction of the steroid. The presence of steroid-metabolizing enzymes in salivary glands has been reported in some studies.44 For example, androstenedione was isolated in an in vitro study in which 14C-labeled T was incubated with human parotid and submandibular salivary glands from women and men.45 Thus, salivary T is not considered to be an accurate marker of circulating free T concentrations.
* Standardization of testosterone and estradiol assays
* Reference ranges of estradiol and testosterone
CONCLUSIONS
Analytical accuracy of hormone assays is critical because inaccurate assays can lead to misdiagnosis or an incorrect assessment of treatment. For many years, E2 and T have been measured predominantly by immunoassays and MS assays, which have very different methodological principles. In recent years, E2 and T have been quantified by direct chemiluminescent immunoassays on automated platforms and by LC-MS/MS assays. Although these assays are used widely for clinical diagnostic testing and in research studies, establishing optimal measurements of these hormones is hampered bytechnical limitations of the assay methods. This is especially true of the chemiluminescent immunoassays, which lack sensitivity for quantifying low serum E2 concentrations in women and specificity for quantifying E2 concentrations following treatment with exogenous estrogens in postmenopausal women. In addition, studiesshow that direct immunoassays should not be used for the measurement of T in women. Without a purificationstep before quantitation of E2 or T by immunoassay, these hormones will not be measured accurately due tocross-reaction of some of their metabolites with the antibody in the assay. Despite the deficiencies in chemiluminescent immunoassays, these assays continue to provide clinically meaningful results, especially at higher concentrations, eg, in assisted reproductive technology protocols where E2 concentrations are elevated. In contrast to immunoassays, a major strength of MS assays is their potential to achieve high analytical specificity and sensitivity, resulting in accurate and precise measurements of E2 and T. In addition, MS assays allow ahigh degree of multiplexing for measuring multiple metabolites in a single run using a relatively small amount of serum or urine. However, MS assays have certain limitations. Accurate and precise measurements requir ethe use of internal standards to monitor procedural losses, which are difficult to prepare. In addition, MS assays are complex, technically demanding, expensive,and require a highly trained technician to operate the instrument
Since MS assays commonly differ by their use of MS and chromatographic equipment, operational conditions,and extraction procedures, the CDC has standardized E2 and T MS assays as well as immunoassays. This is essential to obtain accuracy and uniformity of test results among different laboratories. In addition, the CDC HoSt program provides valuable technical assistance to participants who analyze E2 and/or T in serum samples obtained from the CDC. Also of importance is the establishment of postmenopausal reference ranges for E2 and T by the CDC. These reference ranges are scarce but are essential for providing accurate test results.
