Many practitioners and patients alike are interested in learning more about the differences between dried blood spot testing and other lab testing methods. So we at Rupa Health reached out to Dr. David Zava, a biochemist by trade, Founder and Laboratory Director of ZRT Laboratory, and a pioneer in dried blood spot testing, to get a comprehensive overview of this subject.

Below is an overview of the many benefits and a few disadvantages of choosing dried blood spot testing (DBS) in your practice.

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Why Measure in Blood Spot?

Serum and plasma derived from conventional venipuncture blood draws have been the mainstay for testing hormones, blood lipids, and many other analytes. However, dried blood spot (DBS) is proving to be a simpler and more convenient alternative for testing many of the same analytes present in serum/plasma.

DBS is prepared by a simple finger prick with a disposable lancet and collection of single blood drops onto a specialized filter paper.  Spotted blood is then allowed to dry for several hours, stored, or shipped back to ZRT Laboratory for testing.  Analytes to be tested in the DBS are stabilized by drying the blood, allowing the DBS to be sent by regular mail without coolants, significantly reducing costs.  

DBS collection is more convenient for the patient and the health care practitioner. DBS testing ordered by the practitioner allows the DBS kit to be sent directly to the patient's home. DBS collection by the patient at home circumvents the need to drive to a blood collection center.

Home collection of blood circumvents the need for the collection to be performed by a trained phlebotomist at a clinical laboratory or physician's office. The filter card containing the DBS can be shipped without coolants, reducing weight, saving shipping costs, and providing the convenience of never having to leave home or pay for the extra laboratory costs of shipping a sample on an ice pack, ice, or dry ice. Removing these inconveniences reduces the burdens commonly associated with typical serum or whole blood draws.

Advantages of Testing Analytes in Dried Blood Spot vs. Serum

• Once the DBS card arrives in the laboratory it is labeled with a unique barcode that allows for tracking and punching discs from the blood spots for individual analyte testing.  The barcode significantly reduces human error and is fully automated and driven by the tests defined by the barcode. Working with dried blood on the filter card makes it easier to receive, process, store, and save the sample longer in case retesting or add-on testing is deemed necessary. Venipuncture blood serum/plasma, on the other hand, is discarded immediately after testing the ordered analytes making it unavailable for retesting or add-on testing.
• Analytes tested in DBS are stable for > 1 year when stored at <= -20C and for many years at -80C, making DBS an attractive and superior alternative to serum/plasma for research testing.
• When the DBS card is filled with 12 full single blood spots as many as 20-30+ analytes can be tested by immunoassay and far more by LC-MS/MS (e.g., 23 steroids are tested with only 2 x 6 mm discs punched from 1-2 blood spots). For the variety of different tests performed with one full 12-spot DBS card, this would require multiple tubes of venipuncture blood.
• There is far less risk to the technician handling DBS than liquid blood.  Most infectious pathogens are destroyed by drying blood on the filter card, which significantly reduces the risk of infection for laboratory personnel.
• DBS cards take up much less space and are more convenient and easier to store, retrieve, and process for testing.
• Finger-stick blood represents capillary blood that is transporting oxygen, hormones, and nutrients to tissues that feed the cells of that tissue. Venipuncture blood serum/plasma represents blood spent of oxygen, hormones, and nutrients.
• DBS capillary whole blood provides a more accurate representation of the tissue levels of hormones and nutrients than is venipuncture serum.

What Analytes Can Be Tested Using Dried Blood Spot?

Dried blood spot (DBS) testing has been shown throughout the scientific literature to be applicable to nearly all the analyte categories for liquid blood serum/plasma testing. DBS testing has many advantages over liquid serum and plasma as a matrix for testing various blood analytes, including steroid and peptide hormones, blood lipids, vitamins, essential elements and heavy metals, viral antigens and antibodies, and antibodies to food allergens. DBS tests for these different analytes have been shown in most cases to be quantitatively equivalent to serum.  

DBS vs Serum/Plasma for Detection of Steroid Hormones Used Topically

At ZRT Laboratory, we have found that DBS results are quantitatively equivalent to levels seen in serum/plasma measured by immunoassay, and by LC-MS/MS, in patients NOT on exogenous topical steroid hormone replacement therapy.

In patient’s on topical steroid hormone therapy we and other labs have observed a strikingly higher level of the topically delivered hormone in finger-prick DBS and saliva vs.venipuncture serum/plasma or urine. With topical steroid hormone delivery there is very little increase in the hormone in venipuncture serum over physiological dosing.

In striking contrast with serum and urine, levels in DBS and saliva increase linearly with dosing and reach optimal physiological levels with physiological topical dosing. For practitioners using serum and urine to monitor dosing this has resulted in justifying the use of much higher pharmacological topical hormone dosing. Even at doses 10- to 100-fold higher than physiological amounts fail to achieve physiological concentrations in serum and urine. This is particularly true for topical progesterone.

This unusual phenomenon described above is not unique to topically delivered progesterone and includes all other topically delivered steroids-estrogens, progestogens, androgens, and the adrenal hormones DHEA and cortisol.

We strongly believe that if you are using topical steroid hormones for therapy, the body fluid of choice should be either DBS or saliva, not serum/plasma or urine. It is important to keep in mind that this only applies to topically delivered hormones; when steroid hormones are produced endogenously, serum, urine, saliva, and DBS are acceptable for testing by immunoassay or LC-MS/MS.  The chart below outlines what we have found are the most ideal body fluids to use when testing steroid hormones that have been delivered by different routes of administration.  

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Disadvantages of Dried Blood Spot Testing

DBS testing mostly has advantages but a few minor disadvantages to testing in other body fluids (serum/plasma, urine, and saliva) that are worth mentioning.  

• DBS represents total steroids in the interstitial fluid admixed with capillary blood and not the free fraction of steroids bioavailable to tissues, as with saliva.
• DBS testing of steroids requires more extensive preparation in the laboratory (punching of DBS discs into 96-well plates, extraction of analytes from the dried blood, centrifugation, and more sensitive methods of analysis (e.g. LC-MS/MS) than conventional serum testing with auto analyzers using immunoassays. ZRT Laboratory performs DBS steroid testing only by LC-MS/MS due to the higher nonspecific background that interferes with immunoassays used for testing estradiol and testosterone, present at very low levels in some men and women.  
• Proficiency testing for DBS is limited, but due to quantitative equivalence with serum/plasma when testing endogenously produced steroid hormones can be compared with serum/plasma samples mixed with red blood cells to create DBS controls and calibrators.
• Compliance: Some individuals have an adverse reaction to poking their finger with a needle vs. "look-away" venipuncture at a clinic by a phlebotomist.
• Topical hormones applied with the hands within 24 hr of the finger-stick collection can contaminate the fingertips resulting in false-high test results that are not an accurate representation of capillary/interstitial tissue levels in other areas of the body. Care must be taken to avoid applying topical hormones with ungloved hands or applying topical hormones to the hands, wrists, or lower arms, as this will likely result in accidental contamination of the fingertips used for DBS collection.
• Hormones should not be applied topically with the hands to the upper parts of the body for at least three days before DBS or saliva collection; however, hormones should be used daily as recommended but applied below the waist (e.g. inner thighs) three days before DBS collection.

Summary

A dried blood spot is a form of collection where patients place blood drops on a filter card after a finger prick with a lancet. Once dry, blood spot cards are extremely stable for shipment and storage, and the dried blood format offers excellent correlation with serum tests.

Blood spot is ideal for measuring steroid hormones and other analytes such as insulin, blood lipids, Vitamin D, thyroid hormones, essential and toxic elements. It offers distinct advantages over serum because it eliminates the need for a blood draw – saving patients time and money.

ZRT developed the science for accurately measuring steroid hormones in dried blood spots by LC-MS/MS and is currently one of the only labs to offer this method commercially.