using Mitra with VAMS to monitor steroid hormones

An article first published on July 1, 2020 in the Annals of Clinical Biochemistry by David J Marshall et al from the clinical biochemistry department at Wythenshawe Hospital (South Manchester, UK) validated an LC-MS/MS method from dried Mitra samples for the analysis of three steroid hormones.

The paper, titled "Quantification of testosterone, androstenedione and 17-hydroxyprogesterone in whole blood collected using Mitra microsampling devices" describes a thorough analytical validation comparing dried Mitra microsamples (collected from both EDTA blood and capillary blood) to plasma/serum levels.

To overcome blood plasma/serum partitioning ratios, the group also calibrated their dried blood values to serum/plasma values by measuring hemoglobin (Hb), converting this to a hematocrit (HCT) levels. 

Background on Measuring Steroid Hormones in Blood 

Why measure steroid hormones? 
Hormones are chemical messengers stimulated by the endocrine system to maintain homeostasis. Perturbations in normal hormone levels can be symptoms of a disease or can sometimes be the cause of a disease. There are three classes of hormones. The first two classes are amino acid-derived hormones, such as epinephrine or melatonin; and peptide-hormones, such as insulin and growth hormone. The third class of hormones, based on the cholesterol backbone, are known as steroid hormones. Steroid hormones are, in turn, classified into five groups: glucocorticoids, mineralocorticoids, androgens, estrogens and progestogens.  

Measuring hormone levels allows us to help diagnose and monitor diseases based on changes in normal hormone levels for an individual. The focus of the paper by David J Marshall et al, looked at measuring three steroid hormones (testosterone, androstenedione and 17-hydroxyprogesterone) using Mitra^® devices based on VAMS^® technology. 

Functions and Diseases Related to Testosterone, Androstenedione and
17-Hydroxyprogesterone Levels 
Testosterone (T) is part of the androstane, or androgen, class of hormones primarily involved with the development of the male reproductive system. Testosterone is also produced by females, but at much lower levels. As discussed in the paper, T is important in the diagnosis and monitoring of hypogonadism in males. 

For example, abnormally low levels of the hormone can lead to a number of problems, including delayed onset puberty in younger males. In mature males,  hypogonadism can cause reduced libido and loss of facial hair, as well as reduced muscle mass and bone density, with the latter leading to a risk of osteoporosis.   

A precursor in the biosynthesis of T and other sex hormones, such as estrogen, is androstenedione (A4). Marshall et al comment that measuring an excess of A4 and T in females helps to diagnose androgenic-secreting tumors, polycystic ovary syndrome (PCOS), and Cushing syndrome —- an illness caused by excess Cortisol (a stress hormone formed in the same steroidogenesis biosynthetic network as A4 and T).

Finally, the paper also highlighted the role of 17-hydroxyprogesterone (a glucocorticoid precursor hormone) in helping to diagnose congenital adrenal hyperplasia (CAH). CAH is a group of autosomal recessive disorders that affect development of primary and secondary sex characteristics in children or even adults. Marshall et al commented that “the measurement of serum 17OHP and A4 is recommended for monitoring treatment of CAH.”   

Alternatives to Venipuncture for Monitoring Steroid Hormones  

The study authors emphasized that the primary alternative to venipuncture is conventional dried blood spot (DBS). However, they also noted that due to the nature of differing hematocrit (HCT) levels that vary depending on the size of the blood spots sampled onto a DBS card, the inconsistent concentration of the analytes measured can affect results. Furthermore, the group commented that chromatography on DBS cards “may limit their use for particular analytes.”  

The issues highlighted by the researchers are due to a popular ‘sub-punch’ workflow, whereby dried blood spots on DBS cards are hole-punched as an initial step in extracting samples from DBS cards and filter papers. The reason for such a workflow is that sub-punching DBS standardizes the physical size of blood samples for extraction. However, each sub-punch does not necessarily contain the same volume or concentration of blood. High HCT levels lead to less blood migration on the DBS card and, thus, sometimes lead to positive biases in analytical results (and vice versa for low percentage HCT). 

Moreover, due to the chromatographic effects highlighted by Marshall et al, sub-punch location can also adversely influence results. Employing volumetrically collected microsamples eliminates these issues. For this reason, the research group decided to use volumetric absorptive microsampling via Mitra devices with absorptive VAMS tips for measuring T, A4 and 17OHP to see if a robust validated method could be developed.   

Mitra with VAMS Hormone Study Methods and Findings: 

• The analytical method was developed on an LC-MS/MS platform following FDA recommendations for validation of bioanalytical method. The method was found to be robust and reliable and “satisfied the criteria for all aspects of the validation.” 
  
  
• During the validation, EDTA venous blood (ethically approved surplus) and capillary blood (collected from volunteers) were sampled onto Mitra devices and compared against wet serum samples.
     
• To correct for serum levels, first the Mitra devices (10 µL) were vortexed in deionized water to allow for dissolution of the dried blood. A small sub aliquot was taken and transferred to 384 well plate for hemoglobin spectroscopic measurement using sulfolyser (SLS) reagent. The hemoglobin value was then used to calculate HCT for each sample. The remainder of each sample was then extracted by LLE for quantitative LC-MS/MS analysis of the 3 hormones.
   
• The HCT value, calculated from the spectroscopic measurement, was then used to adjust the dried blood hormone values obtained from the LC-MS/MS. The reason this adjustment was required, was because plasma/serum concentrations for these hormones are often higher than for whole blood. For example, the paper highlighted that, when binding to sex hormone-binding globulin, T is excluded from the red blood cells, thus such an adjustment is required. The study showed that A4 and 17 OHP also was affected by this negative bias and required adjustment due to percent HCT.
   
• To test for stability, the Mitra samples were tested at various conditions, such as long term cryo-storage, and to simulate ‘the journey of the sample.’ Mitra was stored at -20°C for up to 200 days where all three hormones were stable, except for 17OHP, which maintained stability for up to 150 days. All three hormones were stable for 14 days at room temperature, and three days at 37°C.
   
• The group also tested Mitra samples at 60°C but were unable to extract blood from the VAMS tips in this condition. While this study group extracting steroid hormones faced challenges here, another group (Stove et al) has since extracted Thiamine from Mitra samples for up to six days at 60 °C with success. Therefore, microsampling in this condition may require further studies to refine. 
   
• The group found a strong correlation between plasma to EDTA blood on Mitra for all three hormones, when HCT correction was employed. A similar correlation for A4 and 17OHP was observed when serum and capillary blood was collected on Mitra. However, a large negative bias could be seen for T. The researchers postulated that this was not due to the Mitra device, but due to the differences in capillary and venous blood composition.  

Conclusions of Steroid Hormones Study Authors 

Marshall et al concluded that they had successfully validated an LC-MS/MS method for T, A4 and 17OHP and this method was compatible with dried blood samples collected volumetrically from Mitra devices.  Due to good HCT corrected capillary blood correlation of A4 and 17OHP to serum levels, they proposed this approach could be used for monitoring CAH due to 21-hydroxylase activity. However, due to the bias they saw with T for capillary samples, more work would need to be done to “establish Mitra-specific reference ranges for capillary blood.” 

Final Thoughts 

This important work from the team at Wythenshawe Hospital, demonstrated that a robust method could be developed from samples collected using Mitra devices and would be useful in disease monitoring. For example, this approach could be employed in drug trial research into CAH due to 21-hydroxylase activity. A very important finding from the paper is that sample location on a DBS card is important for extracting some analytes. For example, this study demonstrated that T showed a negative bias in capillary blood but not in venous blood compared to standard plasma or serum.  

Although, interestingly, the related hormones, 17OHP and A4 were equivalent independent of sample location.  There are many examples in the literature where other molecules have behaved similarly. Nevertheless, it is always critical to compare venous against capillary when transferring methods from venous obtained blood to capillary collection, as the composition of the blood between the two sites may be different, which will impact the assay bias.  

This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director as curated content. For details about the study reviewed here, please refer to the original article published online in the Annals of Clinical Biochemistry.