Clinical and Laboratory Standards Institute standard NBS01—Dried Blood Spot Specimen Collection for Newborn Screening provides descriptive, stepwise processes and procedures for collecting blood spot specimens for newborn screening. This standard was developed and continues to be updated to promote uniform practices for demographic newborn data and specimen collection. This standard informs and instructs health care professionals who collect and submit dried blood spot specimens on procedures for skin puncture and pain management; application of blood collected by heelstick directly onto the preprinted circles of the filter paper; blood source recommendations and other blood spot specimen collection techniques; filter paper, handling, and mailing package specifications; specimen collection device specifications; handling blood spots collected on filter paper for DNA and/or RNA analysis; and storage and retention of residual specimens. An expanded appendix section provides additional images and descriptions of unacceptable blood spot specimens.

This standard replaces the previous edition of the approved standard, NBS01-A6, published in 2013. Several changes were made in this edition, including: 
• Revising title 
• Introducing easy-to-follow step-action tables that include comments for each action (as applicable), consistent with CLSI's goal to make standards and guidelines more user friendly 
• Adding definitions for check sum character, collection device//specimen collection device (for newborn screening), dried blood spot, expected range, false-negative screening result, false-positive screening result, incision, in-range result, medical device, newborn screening program, newborn screening system, out-of-range result, puncture, recollection, and specimen acceptability 
• Adding information clarifying use of the term "filter paper" as the blood collection (specified filter paper) section of the specimen collection device, which also has a section for recording demographic and other requested information covered in Subchapters 1.1, 3.1, and 3.1.1 and in the Chapter 3 introduction 
• Updating filter paper considerations (see Subchapters 3.2 and 3.2.1) and shelf life (see Subchapter 3.4.4) 
• Adding information on identifying the patient (see Subchapter 4.3) and verifying patient and specimen identification at the time of collection (see Subchapter 4.5) 
• Adding information and references for acceptable blood specimen sources and collection sites (see Subchapter 4.6), including new Figures 2 and 3 and optimal capillary puncture depth (see Subchapter 4.6.1) 
• Adding discussion on preventing specimen contamination and preanalytical DNA analysis considerations (see Subchapter 5.1) 
• Adding information regarding proper documentation, which includes using a procedures manual, confirming patient identification, documenting consent or refusal, and confirming the demographic data and the expiration date on the collection device (see Subchapter 5.2.1.1) 
• Adding an alternative for warming the newborn's heel (see Subchapter 5.2.2.1) 
• Clarifying the benefits of single-use permanently retractable puncture devices for both the newborn and worker safety (see Subchapter 5.2.3) 
• Adding comment on making a single puncture that the specimen collector must not perform immediate repeat puncture at the same site (see Subchapter 5.2.3, step-action table step 8) 
• Adding instructions on postcollection care (see Subchapter 5.2.4, step-action table step 10, and Subchapter 5.3.3, step-action table step 9) 
• Adding information about anticoagulants to heelstick with capillary tube collection and application (see Subchapter 5.3) 
• Updating information on DBS specimen acceptability and added an image of a good-quality DBS specimen in the new Figure 4 (see Subchapter 5.7.3.1) 
• Indicating that records should include documentation showing delivery date and time, as well as the individual who received the specimen (see Chapter 6 introduction) 
• Revising Appendix A to maintain consistency with the updated collection method instructions, which are covered in Chapter 5, and: 
– Adding preliminary precaution steps (see Appendix A, section A1) before site preparation 
– Dividing former sampling techniques section into three sections: 
• Site preparation (see Appendix A, section A2) 
• Puncture (see Appendix A, section A3) 
• Direct application, collection, and preparation for transport (see Appendix A, section A4) – Revising former section on pitfalls to precautions (see Appendix A, section A5) 
• Revised bullets to provide more concise instructions 
• Revising title of Appendix B and replacing previously used figures with new images of individual DBS, each with an explanation of the specimen quality issue shown 
• Updating Appendix C per its source document and clarifying the scope and purpose of this protocol for testing the absorption characteristics of filter paper 
• Adding interferences affecting NBS results to Table D1 in Appendix D: 
– Removing hypothyroidism treated with carbamazepine from the list of maternal conditions 
– Adding the following maternal conditions: 
• Intravenous iodinated contrast administration 
• Glutaric aciduria type I 
• Medium-chain acyl-CoA dehydrogenase deficiency 
– Adding the following newborn conditions: 
• Biliary atresia, septicemia, and trisomies 
• Hemolytic anemia 
• Jaundice 
• Meconimum ileus 
• Renal insufficiency 
• Very low birth weight – Adding the following treatments used in special care baby units/neonatal intensive care units: 
• Cefotaxime 
• Selected antiseizure medications 
• Moving filter paper specifications formerly in Appendix E to Chapter 3, Specimen Collection Device Considerations (see Subchapter 3.1) • Updating Appendix F (formerly Appendix G): 
– Added "Repeat analysis to validate previous NBS results" to list of possible uses for residual DBS specimens that need prioritization

This standard describes the collection of blood specimens for newborn screening (NBS) programs, including equipment, sources of blood, collection sites, and techniques for blood spot specimen collection. This standard is intended to improve and ensure the quality of dried blood spot (DBS) specimens collected from newborns to rapidly identify newborns at risk for some congenital disorders (eg, inborn errors of metabolism, endocrinopathies, immunodeficiency disorders).

NBS01 includes detailed instructions for the preferred method of blood collection (heelstick with direct application) and also provides easy-to-follow illustrated instructions (see Appendix A) for the steps to be taken by personnel who work at facilities other than the NBS laboratory to ensure acceptable-quality blood spot specimens are collected and to prevent the need for specimen re-collection. Poor-quality specimens (shown in Appendix B) may result in false-positive or false-negative NBS test results and/or the inability to screen the baby. The need for re-collection results in additional follow-up, which if not completed in a timely manner, could result in a missed or delayed diagnosis, unnecessary trauma to the newborn, and anxiety to the parents or guardians.

In all NBS programs, results turnaround time is critical if treatments to alter the adverse consequences of a screened disease (eg, irreversible brain damage or death) are to commence in a timely manner. The specimen criteria and handling procedures describe common variances among different sources of NBS specimens that might be tested and the influence of specimen quality on NBS test results and their interpretation.

In addition, this standard specifies:
• Minimum necessary information to provide on or with the specimen collection device

• Requirements for the two collection device components:

– A section for recording demographic and other requested information

– A blood collection (specified filter paper) section for applying and drying the blood drops

• Minimum requirements for the filter paper matrix, including the protocol for testing the absorption characteristics of filter paper (see Appendix C) on which the blood spots are collected

• Acceptable alternative methods and instructions for collecting blood specimens for NBS testing from the following sources:

– Venous blood

– Umbilical catheter blood

– Femoral catheter blood

• Requirements for handling, shipping, retention, and storage conditions for DBS specimens
Intended users of this standard include:

• Hospital personnel, midwives, or other health care workers who collect DBS specimens during the first few days of a newborn's life

• NBS laboratory personnel who perform testing

• NBS follow-up personnel

• Manufacturers who develop testing methodologies and specimen collection devices

• NBS program advisory committee members

This standard identifies steps in NBS program processes for which it is appropriate to develop written documentation, specifically when it benefits the NBS program and the families (eg, obtaining consent for NBS and DBS specimen retention, DBS specimen transport) while recognizing that local rules and regulations must be observed. Additionally, information on long-term storage and retention of residual specimens is included. In addition to specimen quality issues, this standard lists several patient conditions and treatments known to interfere with the reliability of NBS results (see Appendix D). For more specific information about these conditions and treatments for preterm, low birth weight (LBW), or sick newborns, refer to CLSI document NBS03.6 Many aspects of this standard are also appropriate and useful for the collection of DBS specimens used for other reasons, eg, DNA or RNA molecular methods, at-home specimen collection, and a variety of other tests using a similar filter paper matrix. In addition, most elements of this standard are applicable to blood collection on filter paper from fingerstick punctures of infants, children, adolescents, and adults (see CLSI document GP421).7-10 This standard does not discuss:

• Local NBS program requirements (check local regulations and institutional policies)

• Patient registration or examination (test) request and ordering guidance (refer to CLSI documents GP3311 and GP421)

• Local policies on unacceptable specimens