NACB - Scientific Shorts
NACB - Scientific Shorts (formerly NACB Blog)
By Donald H. Chace, PhD, MSFS, FACB

​Collection of a few drops of blood on filter paper from the heel of newborns has been the specimen of choice in neonatal screening laboratories for nearly 50 years.   Robert Guthrie pioneered the use of this sample collection device for detection of phenylketonuria (PKU).  The filter paper, which is prepared from cotton (cellulose) linter, is attached to a form for recording patient information.  The card has historically been called a Guthrie Card or PKU card.   The filter paper is usually marked with dashed circles that define zones for the nursery to apply blood from the heel of newborns.   The volume of a blood-filled circle is around 50-75 µL.   Each card usually has between 4 and 6 circles such that the total volume of blood collected from a newborn is approximately 300-500 µL.  Filter paper for use in newborn screening is an FDA-registered Class II medical device with specifications regarding the volume of blood at 55% hematocrit that will be present in a punched disc of a specific diameter (i.e., specific surface area).  The most common sample sizes obtained for analyses are either a 1/8th- or 3/16th-inch punches, which correspond to blood volumes of 3.4 and 7.7 µL, respectively for a 55% hematocrit standard.
Typically, newborn screening labs are not considered diagnostic laboratories although some diagnostic laboratories do perform newborn testing of dried blood spots.  I have often written on the topic “Screening versus Diagnostics” and it relates to perceived and often real differences between obtaining samples as a fluid in a vial, tube or other container versus a dried specimen.   The issue is not quite that simple so the following discussion is required.  First, the volume of blood per unit area in a dried blood sample (DBS) will vary between newborns due to variations in hematocrit.   Higher hematocrits are associated with higher blood volumes per unit area of the blood spot and are presumably due to less spreading on application.  In addition, lower volumes of blood per punch are present in samples that do not fill the circles and are significantly less than 25 µL. This is presumably due to lack of saturation of the card.  There are other problems associated with the collection of specimens but it is worth noting however that this variation is usually 15% or less, a value often acceptable in many laboratories.  Second, the volume of blood or plasma and urine is very small (<10 µL) as compared to a typical clinical chemistry analysis that utilizes a few hundred microliters.  Therefore methods have to exhibit sufficiently low limits of detection to be able to measure the concentrations of these metabolites.  
What I presented above are some of the limitations often cited for the use of filter paper.  But I do believe they can be overcome for many assays.   The analytical sensitivity issue is overcome by methods with a broad range of sensitivities.   Tandem Mass Spectrometry has worked well in this regard.  The issue of quantification of analytes can be improved by using stable isotopes although it is important to note that these standards are often added to the extraction step of a DBS and not as good as adding stable isotopes to liquid specimens (known as isotope dilution mass spectrometry, IDMS).   One approach that has worked well in newborn screening is to calculate concentrations relative to other metabolites. For example, the ratio of Phenylalanine to Tyrosine (Phe/Tyr) is perhaps a better indicator of PKU and its subsequent treatment monitoring that the concentration of Phenylalanine alone.  It also has less variability due to blood volume.  These ratios, which I call molar ratios, require a non-classical approach for issues such as LODs LOQs etc. There are other advantages to filter paper that are well known such as easier sample storage, reduced shipping cost, improved sample stability, lower collection volumes, and reduced risk of infection that could take several more paragraphs to describe.
Finally, it is worth noting that a few major pharmaceutical research laboratories have begun to investigate the use of dried blood spots and there have been more presentations on the subject recently.  It is likely there will be many new solutions to improve this method of sample collection. The question perhaps should be asked: when, rather than if, clinical laboratories will use dried blood specimens routinely.


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Posted by
On 3/10/2012

Thanks for that comment. I had heard this was being done. Clearly your work shows that DBS are useful in toxicology screening and studies. D.Chace

Posted by
On 3/10/2012

1. You would have to know the hematocrit to correct for variation and I don't think there are any methods that use the same dried blood spot (you would have to collected liquid blood and that defeats the purpose). 2. From a newborn screening perspective, a full circle (the marked dashes) is approximately 75uL. If the blood is well within that zone or outside of that zone, then you are approaching 100 or 50 uL respectively. But the blood volume from an actual punch shouldn't vary substantially within these volumes and close to filling the marked circle. There are manuscripts from the CDC that have been published that describe these variations - check out the CDC website - search for newborn screening or look for papers by B Adams and W H Hannon in ClinChem which will be most helpful to you. One solution, if not newborn screening, is to use a calibrated pipette after collecting in a syringe. If you are doing this you might consider adding internal standard (stable isotope labeled if using MS) to the blood before spotting. 3. Internal standard for DBS varies by metabolites you are measuring. As mentioned above, if using MS then I suggest a stable isotope enriched analogues of your metabolites. If not then use standards typically used in your assay.

Posted by
On 3/9/2012

Labs have been performing lead testing on blood collected from children onto filter paper for several years. I developed a procedure for our GFAA analyzers about four years ago. The test is more challenging for the lab in that it is more time-consuming owing to extraction process, it requires calibrators and controls spotted on filter paper circles and samples are typically run in duplicate (preferably two spots each run a single time). Filter paper extractions cannot be run in the same batch with conventional whole blood samples. For controls, we use selected patient whole blood samples that have been run several time in the conventional mode. We use commercial blood lead calibratores that we reconstitute with sheep red blood cells rather than water. Once spotted they are stable for several months at room temp protected from airborn contamination. In experiments I have done, I have not seen a significant difference when circles are spotted with 2 drops of a sample compared to one drop, even if the second drop is added up to 30 seconds after the first. Crucial to the collection is that the spot is big enough to punch out a circle and that the blood spot saturates the filter paper over the area of the punch-out. The advantages of filter paper are oriented to the clinic. The process involves a finger stick and often includes a finger stick hemoglobin test. Once clinic staff is trained and experienced, they usually find filter paper more convenient than microtainers in terms of handling, storing and transporting. Neil Conway, PhD Cleveland Department of Public Health Cleveland Ohio

Posted by
On 3/8/2012

1. How can we correct for hematocrit variation in samples applied on DBS? 2. Standardization of sample collection on DBS is an issue? What solutions you may have to make sure we can collect 75 uL every time? 3. What internal standard you feel best suited for DBS? Thank you. Majid Moridani, PharmD, PhD

Posted by
On 2/22/2012

1.DBS in Direct to Consumer testing Clearly this is one advantage to filter paper blood spots. Having seen problems in a newborn screening laboratory where the DBS weren't necessarily collected following established protocols (and this was by some experts in nurserys) I worry when consumers perform these tasks. Issues include double spotting, insufficient blood, serum rings, altering the paper by scratching, incomplete dryness.... etc. For very quantitative metabolite assays it could be an issue. 2. Non Cellulose Cards... Interesting... a reduction of Ion suppression should occur in cellulose paper (current paper). An improvement of extraction efficiency is of most interest although for many of the metabolites in newborn screening, that extraction was quite good (most 90% or better). The issue is volume, standardization and quality control... that has to be established. ....Don Chace