Dried Blood Spots (DBS) as an Alternative to Venipuncture Serum for Testing Hormones


By David T. Zava, PhD

Serum vs DBS – A Brief History

Serum/plasma derived by conventional venipuncture blood draw has been the mainstay for testing steroid, thyroid, and peptide hormones, blood lipids, and myriad other analytes associated with the endocrine system.  However, finger-prick blood drops collected and dried on filter paper (Dried Blood Spot, DBS) and used for testing endocrine biomarkers is proving to be a convenient alternative to serum/plasma testing.

Nearly 60 years ago Guthrie and Susi1 first reported on the use of heel-stick neonatal blood drops dried on filter paper for measuring phenylalanine to detect phenylketonuria, a genetic metabolic disorder.  Today finger-prick DBS is proving to be an excellent alternative to conventional venipuncture blood serum/plasma collections for therapeutic monitoring of the clinical markers associated with the endocrine system.2-10 


Advantages of DBS for Testing Endocrine Biomarkers

DBS collection has advantages over conventional venipuncture, the most significant being that the former can be done at home by the patient and shipped directly to the testing laboratory by regular mail without coolants (cold chain), which is required with liquid blood serum/plasma.  Home collection of DBS and the ability to ship the DBS directly to the testing laboratory circumvents the need to drive to a blood collection center or doctor’s office for venipuncture blood collection.  DBS collection at home also allows for collection to occur at the most appropriate time of day and month, which is important when monitoring sex-hormone levels at specific stages of the menstrual cycle, or in individuals monitoring pre- and post-hormone therapies where timing of collection from last use of a hormone to time of collection is important.

DBS collection has also opened doors allowing anthropologists and endocrine researchers opportunities to study populations in remote areas where access to phlebotomists and equipment needed to process (centrifuge), preserve, and ship (coolants for shipment/refrigeration) liquid blood samples are not available. DBS collections also offer advantages for blood collections in the elderly and children where venipuncture is difficult (poor or small veins) or not possible (remote setting).  

Numerous reviews have been published, mostly over the past 20-30 years, on the pros and cons of finger-prick DBS vs venipuncture liquid serum/plasma for testing a broad spectrum of analytes.  The following review references are a handful of those available.  Here I have focused mostly on the references where DBS has been used for testing endocrine biomarkers relative to serum/plasma.3,8-15 

Since Guthrie first recognized the advantages of stabilizing amino acids like phenylalanine in blood by simply allowing the blood to dry on filter paper, myriad other blood markers have been shown to be stabilized in DBS for prolonged periods of time when processed, shipped, and stored at ambient temperature.8,11  Drying of the blood on filter paper to create the DBS simply removes the water, which inhibits oxidation of biomarkers and effectively inhibits enzymes that otherwise degrade these biomarkers in liquid blood serum/plasma if kept at ambient temperature during shipment. Liquid blood/serum requires cooling or freezing to prevent analyte degradation.8,11

The consensus of many researchers who have reviewed scientific literature published on DBS technology is that most biomarkers that are measured in serum/plasma can effectively be measured in DBS with quantitatively equivalent results once the difference in DBS and liquid blood serum derived from whole blood are taken into consideration,2,3,8-19  

In our experience of developing DBS tests over the past 25 years for steroid and peptide endocrine biomarkers,4,5-7,20 essential elements and heavy metals,22 and Covid antibodies,21 we have found that steroid, thyroid, and other peptide hormone endocrine biomarkers quantified in finger-prick capillary DBS are quantitatively equivalent to serum/plasma levels, when validation methods take into consideration the expected average serum/plasma content of whole blood.  However, there are several exceptions where DBS cannot be used to quantify serum biomarkers, or where the levels of hormones in DBS vs serum are very different. 

Perhaps the most significant drawback to testing endocrine biomarkers in DBS is that the amount of serum in the typical 3 mm or 6 mm disks punched from the original DBS is lower than needed for conventional testing by immunoassays.  Testing the very low concentrations of estradiol in men, postmenopausal women, and children, and testosterone in women and children, is more of a challenge than with serum/plasma testing which uses higher volumes to compensate for lower concentrations of estrogens and androgens in these individuals. For example, one 6 mm disk punched from a DBS contains about 12 microliters (µL) of whole blood, about half of which is blood cells and half liquid blood serum/plasma, assuming a hematocrit of about 40-50%. In processing about 6 µL of blood serum from the 6 mm disk, it is necessary to dilute the blood extracted from the DBS disc about 10-fold to enable extraction from the disk and provide ample volume of sample necessary for analyte testing.

For some steroids such as DHEAS and cortisol, and testosterone in men, levels of these steroids are in the ng/ml range and are high enough to accurately measure them by immunoassays using 96 well plates or automated bead assay formats.8 In contrast, measurement of estradiol and testosterone can be a challenge in some individuals with very low levels of these hormones.  Lower analytical sensitivity of tests for some markers can be a limitation to DBS; however, we and others have found that the limitations of analytical sensitivity with steroid immunoassays can be overcome with more advanced and sensitive methods of steroid testing using LC-MS/MS.3,9,11,19  In our laboratory (ZRT) steroids present in DBS are now only measured by fully validated LC-MS/MS methods as we originally reported.20

Many studies have demonstrated that drying blood on filter paper stabilizes most analytes (steroids) and most other small nonpolar molecules rendering them stable for at least a month at ambient temperature.  This is ideal for research participants or for individuals who prefer to collect blood at home, avoiding clinical environments that may increase risk for exposure to pathogens.3,11,21 

As mentioned above and available in references herein, many clinical research studies have shown that levels of analytes in liquid (serum/plasma) and dried blood (DBS) are quantitatively equivalent when corrected for serum/plasma blood volume.  With DBS testing, the assumption is made that the punched disk(s) from the DBS that are used for analysis of biomarkers contain the same volume of liquid blood serum or plasma, which must be corrected as a constant with validation studies to derive a serum/plasma equivalent.  Slight differences are seen when the blood hematocrit falls outside the normal range of about 35-55% or when disks prepared from blood drops are smaller (<20 ul of blood applied to the filter paper) than the recommended full hanging blood drop of 30-50 ul that optimally should be applied to filter paper.9,17,21 Less accurate results are also seen when the blood drop applied to the filter paper is allowed to bleed through the paper to the backside nonabsorbent protective sleeve.9  This is more problematic with 5-spot collection cards where the back protective cover is more difficult to separate from the filter card during blood spot collection, allowing some blood to flow through the filter card onto the backside cover.  DBS collection instructions must be very clear to prevent bleeding of finger-prick blood drops through the filter card onto the back protective sleeve.


DBS and Serum Show Excellent Quantitative Concordance When Testing Endogenous Hormones

In our experience, testing many of the analytes associated with the endocrine system (steroids, thyroid, peptides, blood lipids) as well as essential and toxic elements22 and viral antibodies,21 we have found excellent quantitative concordance of finger-prick DBS with venipuncture serum/plasma results.5-7,20,23-25 While most steroid hormone therapies and different delivery systems for them (oral, troche, topical, transdermal patch, vaginal, subcutaneous/intramuscular injections, pellets, etc.) give more equivalent results when tested by serum/plasma or DBS,4,5 there is one very notable exception—therapy with topical steroid hormones.


Topically Delivered Steroids Don’t Obey the Rules

For reasons that are not fully understood, but well documented throughout the literature, topically delivered steroid therapy results in dose-associated increases in the levels of the supplemented steroid in DBS and saliva with very little to no increase in venous blood levels of the steroid derived by venipuncture6,7 until the dosing rises above physiological levels.  Of interest, and relevant to the efficacy of topical steroid hormone delivery, physiological topical dosing results in physiological levels of hormones in finger-prick capillary DBS, but not venipuncture serum, plasma, or whole blood6,26 derived from venipuncture.  Levels of urine steroid metabolites also do not increase significantly and follow the pattern of serum in that levels of the supplemented steroids don’t begin to rise above their baseline levels until the dosing is within high-physiological to pharmacological range.

Donate to the Townsend Letter

What we find to be universally true for all topically delivered steroids (estrogens, progestogens, androgens, glucocorticoids) in postmenopausal women and older hypogonadal men is that a physiological dose of the topically applied steroid results in an increase from baseline to physiological levels seen in healthy younger individuals at about 12-24 hr post therapy.  In sharp contrast, the same physiological topical dosing has little impact on venipuncture serum and urine levels of steroids, leaving the impression that the steroid is poorly absorbed.


Topical Progesterone at Physiological Dosing in Postmenopausal Women Increases Capillary Blood (DBS) to Peak Physiological Levels Seen at Mid-Luteal Phase

Some of the best examples demonstrating that topical delivery of physiological amounts of steroids results in physiological levels in DBS are research studies exploring the use of topical progesterone.  What we and others find is that a topical progesterone dose of about 20-30 mg, the amount produced by the ovaries during peak of the luteal phase, results in a DBS level of about 10-40 ng/ml at 12-24 hr post-therapy. This dose of progesterone currently is available in many compounded progesterone formulations, as well as OTC products.  With this same topical progesterone dose, no or very little change in serum/plasma or urine progesterone is seen.

In a clinical study6 investigating progesterone levels in saliva, finger-prick capillary blood (DBS), venipuncture serum, and whole venipuncture blood following topical progesterone use, striking differences in body fluid distribution were seen. In this carefully controlled study women applied a high physiological dose (80 mg) of topical progesterone with gloved hands to the inner thighs for 14 days; and on the last day of application, saliva, DBS, venipuncture serum, and venipuncture whole blood were collected.  Blood samples were numerically coded and sent to our laboratory for testing. Testing was performed and the coded results were sent back to the clinical coordinating center for data analysis and correlation with levels in different body fluids.  What our research group showed is that progesterone rose dramatically above baseline several hours later in saliva and DBS but did not increase at all in venipuncture serum or venipuncture whole blood.  That progesterone was not present in venipuncture whole blood, despite having used this dose for 2 weeks prior to testing multiple times over a 24 hr period with the same dose, dispelled the notion that the progesterone was being carried on the red blood cells and from there delivered to tissues.  In fact, in this study we found that venipuncture whole blood had half the value of progesterone because nearly all the progesterone released into the blood-vascular system is carried in the serum/plasma, and not the red blood cells. 

I and others had originally proposed, as an explanation for the wide discrepancy in saliva and DBS vs serum progesterone levels was that the reason progesterone was not detected in serum was due to it being held by the cellular compartment (red blood cells) of blood and removed from serum by centrifugation of blood. This does not appear to be the case since we showed that whole venipuncture blood was lower (about half) than blood serum levels, demonstrating that the much higher progesterone levels detected in finger-prick capillary blood could not be accounted for by binding of progesterone to red blood cells that are removed with clotting and preparation of serum.


Topical Testosterone Therapy in Men and Women Follows the Same Pattern as Topical Progesterone

It is important to recognize that what we see with body fluid distribution of progesterone following topical progesterone therapy is also true for other steroid hormones delivered topically.  For example, topical testosterone therapy also raises DBS and saliva levels of testosterone in a dose-dependent fashion but has little, or much less, impact on serum levels.  WADA (World Anti-Doping Association) researchers investigating topical testosterone abuse in sports27-29 recently published studies on male and female volunteers who used topical testosterone and measured levels in venipuncture serum and finger-prick DBS and saliva.  What these investigators discovered is that topical testosterone users had a much higher (10-20x) level of testosterone in DBS and saliva than in serum.    These results are more evidence that topically delivered steroids are transported slowly throughout the body without entering venipuncture blood, as we have reported for topical progesterone.6,26


Blood or Lymphatic Delivery of Topically Applied Small Non-Polar Molecules to Tissues?

The most logical explanation for topically delivered steroid hormones showing up in saliva, finger-stick capillary blood (DBS), and tissues6,26,30 is that the hormones are being delivered systemically from the site of application to the tissues without moving through the blood vascular system.  As I have discussed in previous publications,6,7,26,31 it is likely that small nonpolar molecules such as steroids, enter the lymphatics present at the surface of the skin and are slowly transported through the lymphatic system to tissues rich in lymphatics such as the salivary glands, tips of the finger, reproductive tissues (uterus, breasts), and brain (glymphatics).  In fact, this may explain why the level of salivary hormones increases more dramatically than the fingertip because the salivary glands have a greater abundance of lymphatics.  It would also explain why the breast tissue, and likely other reproductive tissues rich in lymphatics, respond so well to the protective effects of topical progesterone31 without affecting serum progesterone levels.


First-Pass or No-Pass Effect with Topical Steroid Therapy

That topically delivered steroids are not present in the saliva and fingertip DBS samples until about 2-6 hr after distant application is consistent with slow transport from site of application to lymphatic-rich systemic tissues.  As we reported6,26 in the study with women using topical progesterone daily for 2 weeks prior to measuring progesterone in different body fluids, there was essentially no progesterone in venipuncture serum or whole blood, meaning that there was no “first pass” effect with progesterone, but rather a “no pass” through the liver via the blood vascular system.  This would explain why we see in our clinical testing for DBS and saliva that other hormones such as DHEA delivered topically, but not orally, rise in a dose-dependent fashion without a similar increase in DHEA sulfate or 7-keto DHEA, as measured by LC-MS/MS.  Again, another example of a “no pass” effect with topical steroid delivery. 


Potential for Overdosing When Measuring Serum or Urine with Topical Steroid Hormone Therapy

The novel concept of steroid hormones being delivered through the lymphatics when applied topically has certainly caused considerable confusion among health care professionals using topical hormones, as these results, based on conventional venipuncture serum testing, could be interpreted to mean that topical hormones are poorly absorbed and that higher, more pharmacological doses, are necessary to raise the serum, and presumably, the tissue levels of the supplemented topical hormone.  An alternate explanation, and one that seems more logical based on serum, DBS, saliva, and urine test results and clinical efficacy of low dose topically delivered steroid hormones, is that topical steroid hormone therapy, which increases saliva and DBS levels in proportion to dosing, would seem to be more accurately portraying tissue levels of the hormone than is serum/plasma derived from venipuncture.  It is important to keep in mind that levels of all the steroids and other analytes (thyroid and peptide hormones) we have tested and validated for commercial testing are quantitatively very similar in serum/plasma and in DBS when the hormones are produced endogenously or exogenous steroid hormones are delivered by other means such as intramuscular or subcutaneous injections, pellet, oral, troche/sublingual, or even transdermal patch therapies. 


More Research Needed

Clearly more research is needed to elucidate the mechanism of transport of topically delivered steroids to systemic sites such as the fingertips, salivary glands, reproductive tissues (breasts and uterus), and the brain.  Clinical studies with application of physiological amounts of topical estradiol and progesterone have shown that estrogenic and progestogenic effects can be seen in breast and uterine biopsies post therapy without significantly affecting serum levels.26,31  If the mechanism of lymphatic transport of small non-polar molecules such as steroids from the skin to reproductive and other tissues is accurate,26 then use of serum/plasma and urine testing may be giving the false impression that the patient using topical steroid hormone therapy is underdosed and may lead to unnecessary dose escalation.  It also raises concern for how small non-polar toxins such as pesticides and herbicides, or other non-polar molecules applied to the skin, might enter the body through the skin and be transported through the lymphatics to tissues where they would concentrate and potentially increase risk for dysfunction and cancer of lymphatics and reproductive tissues.32


References

1.  Guthrie R, Susi A. A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics. 1963. 32: p. 338-43.

2.  Santos CM., Abad R, Cua SC, Domingo CF. (2003). Monitoring congenital adrenal hyperplasia using blood spot 17-hydroxyprogesterone assay. Southeast Asian J Trop Med Public Health, 34 Suppl 3, 174-178. https://www.ncbi.nlm.nih.gov/pubmed/15906729

3.  Freeman JD, et al. (2018). State of the Science in Dried Blood Spots. Clin Chem, 64(4), 656-679. https://doi.org/10.1373/clinchem.2017.275966

4.  Kapur, S., Kapur, S., Groves, M., Zava, D. (2010). Cardiometabolic Risk Screening using Simple and Convenient Dried Blood Spot. Preventive Cardiovascular Nurses Association 16th Annual Scientific Meeting, Chicago, IL, April 15-17, 2010 Chicago, IL.

5.  Edelman, A., Stouffer, R., Zava, D. T., Jensen, J. T. (2007). A comparison of blood spot vs. plasma analysis of gonadotropin and ovarian steroid hormone levels in reproductive-age women. Fertil Steril, 88(5), 1404-1407. https://doi.org/10.1016/j.fertnstert.2006.12.016

6.  Du, J. Y., et al. (2013). Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood. Menopause, 20(11), 1169-1175. https://doi.org/10.1097/GME.0b013e31828d39a2

7.  Zava, D. T. (2021). Topical Therapy with Estradiol, Progesterone, and Testosterone and Their Distribution in Saliva, Capillary Blood, Serum, and Urine. Townsend Letter, 31-34. https://www.townsendletter.com/article/450-topical-therapy-estradiol-zava/

8.  Fischer, S., Obrist, R., Ehlert, U. (2019). How and when to use dried blood spots in psychoneuroendocrinological research. Psychoneuroendocrinology, 108, 190-196. https://doi.org/10.1016/j.psyneuen.2019.06.011

9.  Moat, S. J., George, R. S., Carling, R. S. (2020). Use of Dried Blood Spot Specimens to Monitor Patients with Inherited Metabolic Disorders. Int J Neonatal Screen, 6(2), 26. https://doi.org/10.3390/ijns6020026

10.  Campbell, B. C., et al. (2010). Testosterone exposure, dopaminergic reward, and sensation-seeking in young men. Physiol Behav, 99(4), 451-456. https://doi.org/S0031-9384(09)00397-7

11.  Palmer, E. A., Cooper, H. J., Dunn, W. B. (2019). Investigation of the 12-month stability of dried blood and urine spots applying untargeted UHPLC-MS metabolomic assays. Anal Chem, 91(22), 14306-14313. https://doi.org/10.1021/acs.analchem.9b02577

12.  Chace, D. H., De Jesus, V. R., Spitzer, A. R. (2014). Clinical chemistry and dried blood spots: increasing laboratory utilization by improved understanding of quantitative challenges. Bioanalysis, 6(21), 2791-2794. https://doi.org/10.4155/bio.14.237

13.  Delahaye, L., et al. (2021). Alternative Sampling Devices to Collect Dried Blood Microsamples: State-of-the-Art. Ther Drug Monit, 43(3), 310-321. https://doi.org/10.1097/FTD.0000000000000864

14.  Lim, M. D. (2018). Dried Blood Spots for Global Health Diagnostics and Surveillance: Opportunities and Challenges. Am J Trop Med Hyg, 99(2), 256-265. https://doi.org/10.4269/ajtmh.17-0889

15.  Garza, K. Y.,  Clarke, W. (2022). Dried Blood Spots and Beyond. AACC. https://www.aacc.org/cln/articles/2022/september/dried-blood-spots-and-beyond

16.  Henion, J., et al. (2013). Dried blood spots: the future. https://doi.org/10.4155/ebo.13.442

17. Groh, R., Weiss, L. M., Borsch-Supan, M., Borsch-Supan, A. (2022). Effects of spot size on biomarker levels of field-collected dried blood spots: A new algorithm for exact DBS size measurement. Am J Hum Biol, 34(10), e23777. https://doi.org/DOI: 10.1002/ajhb.23777

18.  Tey, H. Y., See, H. H. (2020). A review of recent advances in microsampling techniques of biological fluids for therapeutic drug monitoring. J Chromatogr A, 1635, 461731. https://doi.org/10.1016/j.chroma.2020.461731

19.  Boelen, A., et al. (2016). Determination of a steroid profile in heel prick blood using LC-MS/MS. Bioanalysis, 8(5), 375-384. https://doi.org/10.4155/bio.16.6

20.  Morimoto, L. M., et al. (2018). Neonatal Hormone Concentrations and Risk of Testicular Germ Cell Tumors (TGCT). Cancer Epidemiol Biomarkers Prev, 27(4), 488-495. https://doi.org/10.1158/1055-9965.EPI-17-0879

21.  Zava, T. T., Zava, D. T. (2020). Validation of dried blood spot sample modifications to two commercially available COVID-19 IgG antibody immunoassays. Bioanalysis. https://doi.org/10.4155/bio-2020-0289

22. Zava, T. T. (2017). Dried Urine and Blood Spot Analysis of Essential and Toxic Elements by ICP-DRC-MS with an Emphasis on Inter-Assay Stability of Samples Kept at Room Temperature [Conference Poster]. 69th American Association for Clinical Chemistry Annual Scientific Meeting & Clinical Lab Expo, San Diego, CA.

23.  Zava, D. T. (2007). Topical Progesterone Delivery and Levels in Serum, Capillary Blood, and Tissues. The Script, 14, 4-5.

24.  Zava, D. T. (2019). Topical Delivery of Sex Steroid Hormones and Distribution in Different Body Fluids. Anti-Aging Medical News, Spring, 20-23.

25.  Kapur, S., Kapur, S., Zava, D. (2007). Dried blood spot screening of cardiometabolic risks. American Academy of Clinical Cardiology Annual Meeting, San Diego, July 15-19, 2007. San Diego, CA.

26.  Zava, D. T., Groves, M. N., Stanczyk, F. Z. (2014). Percutaneous absorption of progesterone. Maturitas, 77, 91-92. http://www.elsevier.com/locate/maturitas

27.  Thieme, D., Rautenberg, C., Grosse, J., Schoenfelder, M. (2013). Significant increase of salivary testosterone levels after single therapeutic transdermal administration of testosterone: suitability as a potential screening parameter in doping control. Drug Test. Anal, 5(11-12), 819-825. https://doi.org/10.1002/dta.1536

28.  Polet, M., De Wilde, L., Van Renterghem, P., Van Gansbeke, W., Van Eenoo, P. (2018). Potential of saliva steroid profiling for the detection of endogenous steroid abuse: Reference thresholds for oral fluid steroid concentrations and ratios. Anal Chim Acta, 999, 1-12. https://doi.org/10.1016/j.aca.2017.11.015

29.  Segura, J., Reverter-Branchat, G., Ventura, R. (2021). Dried blood spots, an emerging tool for doping control. Toxicologie Analytique et Clinique, 33(3), S26-S27. https://doi.org/doi.org/10.1016/j.toxac.2021.06.027

30.  Salamin, O., et al. (2021). Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration. Journal of Pharmaceutical and Biomedical Analysis, 204. https://doi.org/https://doi.org/10.1016/j.jpba.2021.114280

31.  Chang, K. J., Lee, T. T., Linares-Cruz, G., Fournier, S., de Lignieres, B. (1995). Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril, 63(4), 785-791.

32.  Zava, D. T., Blen, M., Duwe, G. (1997). Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture. Environ Health Perspect, 105 Suppl 3, 637-645.