The Use of Filter Paper to Collect Blood Samples for Diagnostic Applications
a report by
MichaelA.Harvey
Research and Development, Schleicher and Schuell Inc., Keene, NH, USA.
Collection of blood samples for both genetic and analyte testing at remote sites is of increasing importance. Concerns
about sample stability, safety, reproducibility and problems in shipping must beaddressed. A method that has been
used for a number of years and, has attracted much attention recently, is the collection of blood samples on filter paper.
This method of sample collection has proven to have many advantages over traditional methods. A blood spot dried
onto filter paper is easily shipped through the mail, handled safely and provides an extremely stable analyte
enjoinment and convenient method of storage. As diagnostic procedures expand to include new analytes and
incorporate new methodologies the advantages of this method are becoming more recognized. In 1963 Guthrie first
published data demonstrating the feasibility of collecting neonatal blood samples onto filter paper for PKU testing of
newborns (1-2). This created a system which simplified the collection, storage and shipment of samples to centralized
testing facilities. Since that time blood collected onto filter paper, has been used to acquire samples for testing of
thyroid hormones, antibodies against infectious diseases, therapeutic drug monitoring, drugs of abuse,
hemoglobinopathies, and circulating lead levels (3-4). In recent years an increased demand for sample collection for
genetic analysis has occurred. Collection of blood samples for this type of testing began with the screening of
neonates, for cystic fibrosis (5). It has now been extended to include hemoglobinopathies, infectious disease testing, in
particular for HIV and HCV infection, genetic identification and genetic predisposition to disease (6-10). In addition,
samples other than blood have been collected for genetic analysis. Progress has been made toward paper collection and
storage of buccal, saliva and urine samples. With this expanding array of applications of paper-based collection and
storage, a variety of new requirements and procedures has arisen. I will discuss the properties necessary for a good,
paper-based collection device, some of the applications which have employed this method of sample collection and
some of the procedural approaches that have been used to process dried samples collected on paper. In addition, I will
discuss new matrices which have been developed to address the requirements of new testing techniques. In order to
reproducibly collect a dried specimen the material must have some standard and highly reproducible properties. The
matrix, in addition to being consistent must not bind the analyte of interest nor contribute interferences to the assay.
Analytes must be easily and reproducibly extracted from the collection matrix. There is an increasing need that the
platform be compatible with robotic or high throughput techniques. It must provide a stable environment for storage of
the analyte and it must have an appropriate absorbency to allow it to collect a sufficient volume for the required
testing. In some cases it must act as a volumetric collection device that does not chemically change the sample. One
problem with dried sample collection is a limitation on volume. The sample volume available is limited to the surface
area that can be incorporated into the extraction procedure. This creates an absorbency requirement that the material
must have to accommodate the volume required to test for a particular analyte. A certain volume of buffer is required
to extract a dry sample and this might create a limitation on achieved sensitivity for a given test. The papers that have
been used for this application have been specifically designed and manufactured to meet these requirements. Papers
are manufactured from cellulose, an abundant, naturally occurring polymer. Unfortunately this also means that,
depending on the source of the material, it can contain a wide variation in potential contaminating substances. A
defined and reproducible source of the raw material must be located to make a contaminate-free, reproducible product.
In order for most papers to have strength, additives are generally included in the formulations. For sample collection
applications this is generally unacceptable because such additives may prove to be extractable or may alter or bind the
test analytes. Therefore most collection papers do not contain additives and have a low wet strength.
In the 1960s Schleicher and Schuell began to manufacture a specimen collection paper called 903. This paper is a
highly controlled cotton linter paper that contains no hardeners or additives. The manufacturing of 903 paper is highly
controlled to yield a consistent paper. Water absorbency, serum uptake, the time required to absorb a standard blood
sample and the size of spot created by a standard blood sample are all tested on each lot of paper. To assure its
noninteractive qualities recovery of thyroid hormones (thyroxine) are routinely measured. Initially, blood collection on
filter paper was used primarily for testing for phenylalanine to detect phenylketonuria in newborn infants. The neonatal
screening programmes then extended the list of disorders measured from filter paper blood spots to include
galactosaemia, branched-chain ketonuria, maple sugar urine disorder, congenital hypothyroidism, congenital
hyperplasia, cystic fibrosis and sickle-cell anemia. All of these tests involved measuring analytes or enzymatic
activities present in the blood sample. Procedurally, most of these tests involve punching a circle from the blood spot
and either allowing the analyte to diffuse from the dried blood spot or extracting the circle with a defined volume of
buffer. Since most clinical decisions revolve around determining the concentration of an analyte, it is important that
the concentration in the extraction reflects the concentration present in the original blood sample. Studies have
demonstrated that different lots of 903 paper are consistent enough to accurately reflect the original sample
concentration (11). Paper can be cut or formatted to nearly any size or configuration. This is particularly important in
high through-put or automated testing environment. Paper collection devices have proven convenient for robotic
testing. In 1987, Edward McCabe reported successfully extracting DNA from dried blood spots collected on filter
paper (12). This result, combined with the advent of nucleic acid amplification techniques, made genetic testing from
paper-collected blood spots possible. With these advances neonatal screening laboratories began to add genetic tests
for hemoglobinopathies, Duchenne muscular dystrophy and cystic fibrosis to their list. However the procedures
required to extract DNA reproducibly from paper-collected dried blood spots were technically difficult. Generally
these included digestion with proteases, addition of detergents, chelating reagents and organic extractions. Though
successful, these procedures do not always lend themselves to routine clinical testing. Perhaps an alternative matrix
would have properties more suitable for DNA extraction. One approach to making a blood collection paper that
allowed for simpler preparation of DNA for amplification was to modify the matrix itself. In 1995 we described a
modified 903 paper called IsoCode which required only a simple water elution to generate DNA for amplification from
dried blood spots (13). IsoCode is an impregnated paper which inactivates many of the PCR inhibitors present in blood
samples. When a sample is applied to IsoCode the impregnants in the matrix lyse intact cells, dissociate proteins from
nucleic acids and, as the drying process proceeds, inhibitory substances are fixed to the matrix. A portion of the blood
spot is then chosen, a circle punched and transferred to a tube containing water. Following a brief incubation period at
90ºC, DNA is eluted into the supernatant. This supernatant then serves as a source of template DNA for amplification.
Our studies indicate that approximately 60% of the DNA in a blood sample is eluted and available for amplification
using this procedure.IsoCode has been used to collect blood samples for genetic identification purposes and mutational
analysis for cancer markers (9-10). Work continues to evaluate the effectiveness of this matrix in the diagnosis of
several other disease states. In addition to collecting blood samples on IsoCode, several investigations are ongoing to
evaluate its effectiveness for the collection of oral samples for genetic analysis. In summary, collection of blood
samples on filter paper for clinical testing has proven to be a highly effective means of sample collection and storage.
For years it has been the standard sample collection method in the neonatal screening programmes for a variety of
congenital diseases. More recently there has been a large number of studies demonstrating the efficacy of this method
for collection of samples for infectious disease diagnosis. Specimen absorption onto paper has proven to be an
extremely valuable technique for the collection of samples from remote sites which can then be shipped to centralized
testing sites. A modified paper matrix has been developed which has been designed to have characteristics, which
make extraction of DNA for genetic analysis simpler and more reproducible. IsoCode has proven effective for the
collection of blood samples for both mutational analysis and genetic identification.
Re f e r e n c e s :
1. Guthrie, R., and A. Susi. 1963. A simple phenylalanine method for detecting phenylketonuria in large populations of
newborn infants. Pediatrics. 32(3):338-343.
2. Guthrie, R. 1968. Screening for ‘inborn errors of metabolism’ in the newborn infant-multiple test program. Birth
defects Original Article Series. IV(6):92-98. National Foundation-March of Dimes.
3. Naylor, E. 1991. New technologies in newborn screening. Yale J. of Biol. Med. 64:21-24.
4. Yee, H.Y., K. Srivuthana, R. Elton, K. Bhambhani, and R. E. Kauffman. 1995. Capillary blood collection by
paper for lead analysis by graphite furnace atomic absorption spectrometry. Microchemical Journal. 52(3):370-375.
5. Prior, T.W., W.E. Highsmith, Jr., K,J. Friedman, T.R. Perry, G. Scheuerbrandt, and L.M. Silverman. 1990. A model
for molecular screening of newborns: simultaneous detection of Duchenne/Becker muscular dystrophies and cystic
fibrosis. Clin. Chem. 36 (10): 1756-1759.
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Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening.
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surveillance tool for molecular epidemiology. AIDS Res. Hum. Retroviruses 12:1435-1441.
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10. Struewing, J.P., P. Hartge, S. Wacholder, S. Baker, M. Berlin, M. McAdams, M.M. Timmerman, L.C. Brody,
M.A. Tucker. 1997. The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi
Jews. N Eng. J. Med. 336, 1401-1408.
11. Slazyk, W.E., D.L. Phillips, B.L. Therrell, Jr., and W. H. Hannon, 1988. Effect of lot-to-lot variability in filter
paper on the quantification of thyroxine, thyrotropin, and phenylalanine in dried-blood specimens. Clinical Chemistry.
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filter paper blotters: potential applications to newborn screening. Hum. Genet. 75:213-216.
13. Harvey, M. A., T. King, and R. Burghoff. 1995. Impregnated 903.