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Filter paper as a blood sample collection device for newborn screening.

Newborn screening (NBS) [3] has been acclaimed by the Centers for Disease Control and Prevention as one of the most successful public health programs of the 21st century (1). In 2013, we celebrated the 50th anniversary of NBS, and these 50 years were marked by many successes. An estimated 200 million newborns in the US have been screened during that period for at least 1 disorder, phenylketonuria. Today's screening effort detects > 10 000 newborns with certain heritable disorders from the 4 million screened annually. Most of these disorders cause severe, nonreversible harmful effects if not identified and treated before the onset of symptoms (2). NBS programs have evolved as new technologies have been introduced from covering 1 disorder to presently 33 recommended disorders in the US (3, 4).

All NBS programs in the US collect blood samples on US Food and Drug Administration--cleared filter paper collection devices (5). Only 2 commercial sources are presently approved for use in device production and dried blood spot (DBS) collection. The filter papers, Whatman Grade 903 and Ahlstrom Grade 226, are made from high-purity cotton linters and manufactured to yield accurate and reproducible blood samples according to the Clinical and Laboratory Standards Institute's specifications (NBS01-A6) (5). Under controlled conditions, the filter paper blood collection device for NBS can achieve the same level of precision and reproducibility expected from clinical methods that typically use blood collected in vacuum tubes or capillary pipettes (6). Unlike these liquid collection devices, filter paper exhibits different levels of imprecision, but these can be characterized and then harmonized to minimize the variation it contributes to measurements (6).

Simplicity of collection, transport, and storage makes DBS samples an economical preference for many clinical applications. However, poor-quality filter paper and improperly collected DBS may significantly alter the performance of NBS, yielding poorer test results, the risk of missed cases, and delays in testing, each creating the need for a replacement sample and a possibly delayed diagnosis. Delays in the screening process can cause issues with the achievement of desired criteria for turnaround time, i.e., time from sample collection to result reporting, and ultimately to diagnosis and start of treatment (5).

It was not until filter paper became widely used in population-based screening that the routine evaluation of filter paper itself became necessary along with establishing defined performance parameters. In late 1970s, with expansion of NBS to include congenital hypothyroidism using the same sample as that for phenylketonuria, improved quantitative performance from the paper was required (7). It became evident to the screeners that a standardized protocol to routinely monitor and evaluate the quality of filter paper within and among manufactured lots was essential to ensure consistency of quantitative assay performance. The manufacturers of filter paper demonstrated that they could produce papers that met the established performance criteria (5). Critical to proper and effective use of the filter paper is ongoing assessment and evaluation of new production lots relative to previous lots by an independent laboratory to ensure that all lots meet the expected criteria before they are released to users (8). This independent laboratory also troubleshoots problems with filter paper issues identified with individual lots by users. To ensure consistency among production lots, the following parameters are monitored: absorption volume, absorption time, physical appearance, and homogeneity. These data are available to users on request (5, 8). The punch from the sample is a volumetric measurement for an analytical method, and thus a high degree of uniformity is essential to minimize variance from lot-to-lot filter paper transitions for calibrators, QC materials, and unknown DBS samples, including the consistent collection of equivalent-size saturated bloodspots (5, 6, 9). Because many nonevaluated filter papers exist in the worldwide market place, caution is advised in the selection of filter paper for sample collections (9).

When using blood samples collected on filter paper, the numerous associated variables are complex. Concerns include sample volume, humidity, hematocrit, chromatographic effects (homogeneity), paper compression, specific analyte recovery, elution efficiency, blood sample source, anticoagulant, and filter paper performance characteristics (5, 8, 9). Questions regarding DBS samples often concern how best to control and minimize the inherent variability. Other issues include the best location for obtaining the optimal sample punch from the spot related to complete absorption and chromatographic effects, the size of sample applied to the filter paper (variations in analyte concentration dependent on sample volume), and the consistency of filter paper characteristics. Random sampling errors related to small testing aliquots from paper punches are also an issue (9). Minimizing the influence of these variables on assays is difficult, especially in uncontrolled sample collection sites, thus making DBS ideally suited for screening assays with established cutoff values that capture the overall influence of all types of variance contributions.

In this issue of Clinical Chemistry, George and Moat (10) describe a comprehensive evidence-based investigation of the impact of DBS variance factors on the detection of 9 disorders. They report that a lack of published evidence exists regarding minimum quality for acceptance criteria for heel-stick blood samples collected for NBS. Although visual examples are available for the types of poor and questionable quality samples, the magnitude of contributions to erroneous screening results is unknown for their multianalyte test panel. In addition, the authors indicate that previous studies to measure the impact of punch location within the bloodspot included only a few analytes, i.e., the influence of chromatographic spreading for each analyte (8).

Because of the minimum data available to support sample quality decisions, the investigators set out to gather evidence with controlled laboratory studies (Grade 903 filter paper) and simulated poor-quality DBS samples for the 9 analytes measured in the NBS programs of the U.K. They measured contributions of a selected set of variables for 9 analytes, including punch location within the spot, compression of spots, blood volume applied to the paper, and heterogeneity of the bloodspot. The analytical impact was unknown for each poor sample category for all analytes in their test panel. These authors wanted to establish a set of supported criteria for acceptance and rejection of collected blood samples on the basis of analytical evidence for each member of their test panel. Lack of consistency in application of the sample quality criteria among the different screening laboratories was partially caused by no appreciation of potential problems produced by analysis of poor-quality samples. The rejection of poor samples based on sound scientific evidence should lead to consistent practice in policies, and requests for repeat blood collections would be minimal. They examined a wide range of known variables that could possibly produce undesirable analytical outcomes, i.e., false-negative (missed cases) and false-positive results. The study did not examine the influence of hematocrit on sample quality. However, other investigators have previously reported on hematocrit effects (8, 11). One variable not considered or discussed in their studies is the significance of bloodspot collection in the field when collected blood clots into the filter paper during collection and then dries. This variable is nearly impossible to examine but may alter the measured outcomes from simulated evaluations for DBS variables.

Poor-quality samples are detrimental to any analytical method and must be eliminated or minimized. In NBS, poor-quality DBS samples necessitate the collection of another sample, thereby impeding the timely diagnosis and treatment of identified cases. The collection of an additional DBS sample requires another heel stick, causing trauma to the newborn and creating anxiety for the family. The acceptance and rejection of samples require the establishment of defined parameters for reliable application by programs; in many situations, however, the analytical evidence for sample rejection is not known for each analyte. Nonetheless, if any 1 analyte in the panel demonstrates problems with a questionable sample type, all such samples should be rejected even if the deemed poor-quality sample is not a problem for other analytes in the test panel. NBS uses analytical procedures with high sample throughput, high sensitivity, and excellent specificity, so any additional steps added to the logistics for correction of a variable must not impede the screening process.

All NBS laboratories attempt to minimize the contributions of all variables associated with the DBS sample by implementing steps to control them to the extent possible, e.g., size of the printed blood collection circle, drying time, elution times, and consistency of the filter paper. The Standard for Blood Spot Collection for Newborn Screening (5) developed during a period of >30 years of routine review and revisions by different expert panels every 5 years was in the reference list of the George and Moat article but apparently not used in focusing the studies or discussions. This standard provides a visual listing and description of good- and poor-quality samples. It also discusses the consequences of filter paper compression, evaluation of filter paper lots, and other variables requiring control to ensure the collection of the highest quality samples. The decision cutoff value established for detection of presumptive cases for each disorder is a mean of thousands of routinely collected, high-quality bloodspots, so the working cutoff contains all variables encountered in sample collection.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.


(1.) Centers for Disease Control and Prevention. Ten great public health achievements-United States, 2001-2010. MMWR 2011;60:619-23.

(2.) Carreiro-Lewandowski E. Newborn screening: an overview. Clin Lab Sci 2002;15:229 38.

(3.) Watson MS, Mann MY, Lloyd-Puryear MA, Rinaldo P, Howell RR. Newborn screening: toward a uniform screening panel and system. Pediatrics 2006;117:296-307.

(4.) Health Resources and Services Administration. Summary of nominated conditions to the Recommended Uniform Screening Panel (RUSP). http://www.hrsa. gov/advisorycommittees/mchbadvisory/heritabledisorders/nominatecondition/ reviews/nominatedconditions.pdf (Accessed December 2015).

(5.) CLSI. Blood collectionon filter paper for newborn screening programs; approved standard-sixth edition. Wayne (PA): CLSI; 2013. CLSI document NBS01-A6.

(6.) Mei JV, Zobel SD, Hall EM, De Jesus VR, Adam BW, Hannon WH. Performance properties of filter paper devices for whole blood collection. Bioanalysis 2010;2:1397 403.

(7.) Dussault JH, Laberge C. Thyroxine (T4) determination in dried blood by radioimmunoassay: a screening method for neonatal hypothyroidism. Union Med Can 1973;102:2062-4.

(8.) Mei JV, Alexander JR, Adam BW, Hannon WH. Use of filter paper for the collection and analysis of human whole blood specimens. J Nutr 2001;131:1631S-6S.

(9.) Hannon WH, Therrell BL Jr. Overview of the history and applications of dried blood samples. In: Li W, Lee M, editors. Dried blood spots: applications and techniques. Hoboken (NJ): Wiley & Sons; 2014:3-15.

(10.) George RS, Moat SJ. Effect of dried blood spot quality on newborn screening analyte concentrations and recommendations for minimum acceptance criteria for sample analysis. Clin Chem 2016;62:466-75.

(11.) Holub M, Tuschl K, Ratschmann R, Strnadova KA, Muhl A, Heinze G, Sperl W, Bodamer OA. Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry. Clin Chim Acta 2006;373:27-31.

Donald H. Chace [1] * and William H. Hannon [2]

[1] PediatrixAnalytical, Centersfor Research, Education and Quality, Sunrise, FL; [2] Independent consultant, CDC Foundation, and Retired from Newborn Screening Branch, CDC, Atlanta, GA.

* Address correspondence to this author at: Pediatrix Medical Group, 1301 Concord Terrace, Sunrise, FL33323. E-mail

Disclaimer: The findings and conclusions in this report are solely those of the authors and do not necessarily represent the positions of Pediatrix or the CDC Foundation.

Received December 16,2015; accepted December 18,2015.

Previously published online at DOI: 10.1373/clinchem.2015.252007

[3] Nonstandard abbreviations: NBS, newborn screening; DBS, dried blood spots.
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Title Annotation:Editorials
Author:Chace, Donald H.; Hannon, William H.
Publication:Clinical Chemistry
Article Type:Editorial
Date:Mar 1, 2016
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