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OVERVIEW OF THE HISTORY AND APPLICATIONS OF DRIED BLOOD SAMPLES

W. HARRY HANNON AND BRADFORD L. THERRELL, JR

1.1 HISTORY

“Everything should be made as simple as possible, but not simpler.”

Albert Einstein (1879–1955)

For almost 100 years, the simple concept of applying biological fluids to filter paper, drying, transporting to a laboratory, and taking aliquots has been adapted into a variety of innovative methods to generate a suitable specimen and sampling matrix for analytical testing. These methods for collecting blood and other biological fluids created a novel way to sample biological fluids and are responsible for initiating the first interest in analytical micromethods (Schmidt, 1986).

Filter paper sampling greatly simplified blood sample collection, handling, and storage over other methods in use at the time. Over the years, the availability of analytically acceptable dried blood spots (DBSs) has significantly impacted a variety of fields of study including newborn screening (NBS); epidemiology (field testing); infectious diseases; environmental research; forensics; therapeutic drug monitoring; illicit drug analysis; toxicology; and toxico- and pharmacokinetic studies of drugs and candidate drugs. Filter paper samples, although conceptually simple with few anticipated matrix effects, have resulted in many unexpected analytical complexities. The utility and acceptance of DBSs have increased over the last 30 years primarily because of concerted efforts to control, minimize, and eliminate these analytical variations.

Perhaps the earliest reference to blood collected on paper can be seen in the well-preserved Mayan murals of Bonampak, Mexico, dated 780AD. One part of the mural shows women pricking their tongues, fingers, and lips while letting the blood drops collect on paper that is then placed in a container and burned to summon the gods. Another section of the illustration portrays a child spreading his fingers with one finger being pricked for blood collection on paper. A cup on one end on the table appears to contain smoldering blood spots. A row of blood-spot images is depicted on the table's edge (Miller, 1995).

With his modification of the Wassermann test in 1911, Noguchi reported using hemolytic amboceptor (old term for hemolysin/antibody) serum absorbed onto filter paper to increase the stability of this reagent for his complement fixation method for syphilis (Noguchi, 1911). But the first credit for using filter paper for specimen collection is attributed to Bang, who reported its use in this manner for an analytical method in 1913 (Bang, 1913). His ingenious approach introduced a method for absorbing blood onto filter paper, drying the spots, and then determining glucose concentrations from the eluate. He first mentioned his specimen collection method in 1907, although it was not published until 1913 (Schmidt, 1986). His method for blood sugar analysis was both practical and reliable. Bang introduced the use of micro-samples of blood (about 100 mg) absorbed into prewashed and dried filter paper and weighed on a balance to measure the sample aliquot size. This sampling and testing technique was designated “Bang's Method” (Van Slyke, 1957; Schmidt, 1986). Bang also performed Kjeldahl nitrogen/protein determinations with a filter-paper specimen-based micromethod. Ivar Christian Bang has been declared the “founder of modern clinical microchemistry” (Schmidt, 1986).

In 1924, Chapman reported using several varieties of absorptive papers in his studies and noted that “any good grade of a nonalkaline filter paper of low ash content and good absorption power may be used” (Chapman, 1924). He typically used Schleicher and Schuell No. 595 paper but reported that “Whatman No. 3 paper was found to be most satisfactory for the Wassermann test.” Chapman conducted a series of investigations to determine whether patient's blood could be collected on filter paper, dried, and then used in the complement fixation test for syphilis. He used DBSs for the following reasons: (1) less blood was necessary (especially important for children); (2) blood collection supplies were simple and inexpensive; (3) risks of specimen spoilage (by bacterial contamination) or hemolysis could likely be eliminated; and (4) specimen preservation for long periods of time with little deterioration would be possible. Blood was absorbed onto filter paper strips (about 4 in.), dried, and then cut into smaller pieces for elution and analysis. Chapman also noted that blood collected on filter paper and dried could be kept for at least 1 month with little deterioration of its complement fixing power (Chapman, 1924).

By 1939, filter paper was more commonly used as a transport medium for blood samples for serological examination (Zimmermann, 1939). Different types of filter paper were available and recommended or preferred by different investigators. For example, one investigator declared that only Canson 435 blotter paper was usable for his studies, while electrophoresis paper and membrane filters were not suitable (Vaisman et al., 1963). Interestingly, serum collected on filter paper was thought to be less satisfactory than blood. Additionally, efficient extraction techniques were considered to be problematic, so elution methods were recommended. Zimmerman evaluated different blotter and filter papers to determine the best product for his test. These papers included Canson 435 blotting paper and several filter papers: Delta No. 310, Schleicher and Schuell No. 589, and Whatman No. 1 (Zimmermann, 1939). He used a sharp hollow pipe to punch 15 mm paper circles for sampling. The amount of serum and whole blood in a sample was determined by weighing. Samples were declared dry after 2 hours of air drying at ambient temperature. Zimmerman also investigated the impact of different solutions, temperatures, and elution times on DBS elution efficiency (Zimmermann, 1939). The best elution was reported to be obtained from phosphate-buffered saline at pH 7.2 when eluted for 2 hours at 37°C. All papers studied were suitable for transporting dried blood and serum. Consequently, the paper with the smallest variation in the absorbed quantity of blood and serum (determined by weighing dried paper punches before and after soaking in blood or serum) was preferred (Zimmermann, 1939).

In 1950, Hogan noted that “A simpler method of collecting blood would be useful for diagnostic activities in general and would be particularly desirable for the diagnosis and treatment of congenital syphilis” (Hogan, 1950). “Indeed the nonavailability of such a simple test has hampered congenital syphilis control programs” (Hogan, 1950). He described a syphilis test that used whole blood from a finger prick collected on filter paper and dried (Eaton and Dikeman No. 613). Strips of paper were saturated with blood from a finger stick (heel, toe, or ear lobe puncture in infants and children) and the strips were air dried on a clean flat surface. Small squares were cut from the dried filter paper strips and microscopically analyzed. This procedure avoided jugular punctures in infants and children—the standard blood specimen collection procedure at the time. Although the new DBS test was not considered as “good” (sensitive) as the standard liquid serum serologic test, it fulfilled an apparent need (Hogan, 1950). The filter paper method was particularly desirable when screening children for congenital syphilis, especially for mass testing programs and home collections. The finger prick procedure quickly overcame parents' objections to jugular punctures on infants and small children. Nevertheless, some investigators continued to downplay the use of DBSs because of the lower test sensitivity when compared with liquid samples in standard laboratory tests and suggested using these DBSs only when no other specimen collection method was possible (Freeble and Orsburn, 1952).

Filter paper techniques for collecting whole blood and serum for studies on eastern equine encephalomyelitis were reported in 1957. However, the reporting investigators found that hemolytic eluate from the dried blood samples impeded readings on samples with low titers for complement fixation reactions (Karstad et al., 1957). They determined that paper discs made of highly absorbent, commercially available white paper were equivalent to discs cut from white blotting paper. Schleicher and Schuell filter paper was reported to be superior in uniformity and tensile strength when wet. Blood samples were collected from birds and horses, and 12.5 mm discs of the type used for antibiotic sensitivity tests were used. Serum was dropped onto the discs until they appeared saturated. Discs of 12.5 mm and 15 mm diameter were found to absorb 0.16 mL and 0.20 mL serum, respectively, and overnight elution was reportedly most effective (Karstad et al., 1957). Adams and Hanson found that exposure of paper discs to 56°C for 1 hour or exposure to 37°C for 7 days resulted in no appreciable titer loss for neutralizing antibodies; and therefore, the technique was declared valuable for epizootiological studies (Adams and Hanson, 1956).

Early methods to obtain DBS aliquots relied on scissors, copper tubes, or precut paper discs. In 1957, the first use of a paper-hole punch device for collecting aliquots was reported. “The filter paper strip is dipped into the blood and allowed to saturate half of the filter paper strip, dried at ambient temperature and then one-quarter inch punches are taken from the dried-blood tip of the paper strip with an ordinary ticket hole...