‘Noise’ with mass spectrometry warrants caution when measuring hormones during menopause
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Clinicians using conventional mass spectrometry to measure hormone levels should use caution when assessing data from postmenopausal women, when very low estradiol levels are difficult to measure accurately, according to a speaker.
Accurate measurement of very low circulating estradiol levels in postmenopausal women is important to investigate sex steroid action in target tissues, Richard J. Auchus, MD, PhD, professor of pharmacology and internal medicine in the division of metabolism, endocrinology and diabetes at the University of Michigan, said during an online presentation at the virtual North American Menopause Society annual meeting. However, immunoassays are inaccurate, and conventional mass spectrometry-based measurement is often too insensitive at typical postmenopausal levels of estradiol, which can run below 5 pg/mL.
“When looking at low values like this, we must be cautious with the interpretation of the data,” Auchus told Healio. “Most postmenopausal women are down around the 15 pg/mL area, so it is going to be difficult to draw conclusions for what estradiol is doing down at those levels without using better methods.”
Pros, cons of mass spectrometry
Mass spectrometry — an analytical technique that measures the mass-to-charge ratio of ions — has several advantages when measuring serum samples, Auchus said. Mass spectrometry tends to eliminate immunoassay cross-reactivity that can invalidate the low levels measured during menopause, and multiple steroids can be simultaneously measured with a single sample, improving sensitivity.
The technology also brings disadvantages, Auchus said: It is expensive, technically demanding, and the instruments, on the best days, are fragile.
“Mass spectrometry is not a turnkey method,” Auchus said in an interview. “It is real people in the lab, running experiments on a day-to-day basis, getting funny results, and trying to repeat them again. Maybe we need to change the column, maybe we need to clean the instrument or replace a part, or run some solvent through it over the weekend to clean it up. That is why you cannot necessarily get results one day. It is not the sensitivity that limits you. It is the noise that is the problem.”
Mass spectrometry used today is based on a quadrupole design — four metal rods with direct and alternating currents tuned to create an oscillating magnetic field, Auchus said. Ions that come in with the right mass-to-charge ratio will oscillate through the quadrupole and go on to the next chamber; all other ions will be expelled through the sides of the quadrupole.
“This is how we chose the ions we want to measure,” Auchus said.
In measuring samples from the Study of Women’s Health Across the Nation (SWAN) study, a longitudinal study of women across the menopause transition that utilized immunoassay, researchers took several steps to measure estradiol levels more accurately among women using mass spectrometry. Steps included an increase in serum volume (extracting 0.2 mL), which was resuspended in a smaller volume and then injecting a large aliquot, allowing a sixfold increase in sensitivity over a pilot method, Auchus said. Researchers also used a longer column and slower flow rate, optimized the source for estradiol and used scheduled isolated time segments to improve signal-to-noise.
“Other groups have developed high-sensitivity estradiol methods using derivatizing reagents, but we run it as straight estradiol,” Auchus said.
In assessing approximately 400 SWAN samples, researchers observed correlation between the immunoassay results and the mass spectrometry results at high levels of estradiol; however, as estradiol levels declined, researchers noted a deviation in the curve, with the immunoassay breaking down at about 20 pg/mL.
“The correlation was excellent until you got down to the very low levels, and then we found that the mass spectrometry assay was reading down to roughly 3 pg/mL, but there was a gradual deterioration of the correlation,” Auchus said. “When you got down to about 15 pg/mL, it started to deteriorate, even though the assay was said to read down to 6 pg/mL.”
Average menopausal testosterone levels are similarly not accurate when measured using immunoassay, he said.
“In reproductive-aged women, estradiol immunoassay is fine, but in the menopausal women, it is not reliable,” Auchus said.
Use a conservative number
Auchus said the discrepancies raise the question, “What is the limit of quantification in your assay?’” The answer is not simple, he said.
“There is not a straight answer for that because it depends on the day and it depends on the samples,” Auchus told Healio. “You would think that human serum, even though it is highly complex, would have the same major components for most people. There could be some operator error, there could be variability in sample preparations. Some just have more noise in the mass spectrometry range where we are trying to look than others.”
Rather than trying to “dig the values out of the noise,” Auchus said his laboratory does not report a level as measurable if there is too much noise relative to the signal. If someone wants to give a limit of detection, they use a conservative number that can be relied on most days, he said.
“This is something that is difficult for people to get their head around,” Auchus said. “They may think the machine operates the same way every day and with every sample. That is not the case. People need to be aware of this.”
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Auchus RJ. Estradiol and estrone: Changing ratios after menopause. Presented at: North American Menopause Society annual meeting; Sept. 28-Oct. 3, 2020 (virtual meeting).
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